Structured Review

Cell Signaling Technology Inc rabbit anti akt
Loss of FlnB induces Cdk1 activity changes through β1 <t>integrin-Pi3k/Akt</t> pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p
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1) Product Images from "Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling"

Article Title: Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0089352

Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p
Figure Legend Snippet: Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

Techniques Used: Activity Assay, Immunostaining, Knock-Out, Western Blot, Activation Assay, Incubation, Laser Capture Microdissection

2) Product Images from "Small Molecule Screen Reveals Regulation of Survival Motor Neuron Protein Abundance by Ras Proteins"

Article Title: Small Molecule Screen Reveals Regulation of Survival Motor Neuron Protein Abundance by Ras Proteins

Journal: ACS chemical biology

doi: 10.1021/cb300374h

Increased Ras signaling upregulates SMN protein abundance. a) Western blot analysis of #9677 cells treated with 10μg/ml cuspin-1, harvested at indicated time points and blotted for p-Erk, total Erk, p-Akt, total Akt and tubulin. Graph shows quantification of phospho-protein to respective total protein ratios, normalized to tubulin as loading control. Data represents the average of duplicates ± standard deviation. b) Western blot analysis for NRas, p-Erk, SMN and tubulin in #3813 SMA patient fibroblast cells transduced with either empty vector or constitutively active NRas G12D viral plasmid. Graph shows fold increase of SMN compared to vector-only control, normalized to tubulin as loading control. c) Comparison of the engineered tumor cell line BJeLR (eLR), expressing HRas V12 , versus the parental line (eH) and the isogenic line BJeHLT (eHLT) which contains all genes ectopically expressed in BJeLR with the exception of HRas V12 . Samples were blotted for SMN and several control proteins. Graph shows the fold increase of SMN in BJeHLT and BJeLR compared to BJeH, normalized to actin as loading control. Data represent average of duplicates ± standard deviation.
Figure Legend Snippet: Increased Ras signaling upregulates SMN protein abundance. a) Western blot analysis of #9677 cells treated with 10μg/ml cuspin-1, harvested at indicated time points and blotted for p-Erk, total Erk, p-Akt, total Akt and tubulin. Graph shows quantification of phospho-protein to respective total protein ratios, normalized to tubulin as loading control. Data represents the average of duplicates ± standard deviation. b) Western blot analysis for NRas, p-Erk, SMN and tubulin in #3813 SMA patient fibroblast cells transduced with either empty vector or constitutively active NRas G12D viral plasmid. Graph shows fold increase of SMN compared to vector-only control, normalized to tubulin as loading control. c) Comparison of the engineered tumor cell line BJeLR (eLR), expressing HRas V12 , versus the parental line (eH) and the isogenic line BJeHLT (eHLT) which contains all genes ectopically expressed in BJeLR with the exception of HRas V12 . Samples were blotted for SMN and several control proteins. Graph shows the fold increase of SMN in BJeHLT and BJeLR compared to BJeH, normalized to actin as loading control. Data represent average of duplicates ± standard deviation.

Techniques Used: Western Blot, Standard Deviation, Transduction, Plasmid Preparation, Expressing

3) Product Images from "Angiopoietin-1 protects myocardial endothelial cell function blunted by angiopoietin-2 and high glucose condition"

Article Title: Angiopoietin-1 protects myocardial endothelial cell function blunted by angiopoietin-2 and high glucose condition

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2010.183

Changes in Tie-2/Akt/eNOS signaling pathways under HG conditions. (A) Tie-2 phosphorylation was decreased under HG conditions in MHMECs ( n =3). (B) HG inhibited phosphorylation of Akt and eNOS in MHMECs compared with NG groups ( n =5). (C) Ang-1 increased Tie-2 phosphorylation in MHMECs, which was dampened under HG conditions ( n =3). b P
Figure Legend Snippet: Changes in Tie-2/Akt/eNOS signaling pathways under HG conditions. (A) Tie-2 phosphorylation was decreased under HG conditions in MHMECs ( n =3). (B) HG inhibited phosphorylation of Akt and eNOS in MHMECs compared with NG groups ( n =5). (C) Ang-1 increased Tie-2 phosphorylation in MHMECs, which was dampened under HG conditions ( n =3). b P

Techniques Used:

4) Product Images from "Expression of HER-2 in MCF-7 breast cancer cells modulates anti-apoptotic proteins Survivin and Bcl-2 via the extracellular signal-related kinase (ERK) and phosphoinositide-3 kinase (PI3K) signalling pathways"

Article Title: Expression of HER-2 in MCF-7 breast cancer cells modulates anti-apoptotic proteins Survivin and Bcl-2 via the extracellular signal-related kinase (ERK) and phosphoinositide-3 kinase (PI3K) signalling pathways

Journal: BMC Cancer

doi: 10.1186/1471-2407-8-129

Signaling pathways involved in survivin regulation in HER2 over expressing MCF7cells . A- HER2 induced MCF7 cells were incubated for 48 hours with specific inhibitors of PI3K (10 μM LY294002) AKT1/2/3 (15 μM AKT IV Inhibitor) and ERK1/2 (10 μM U0126) or without inhibitor (control) and analyzed by western blotting for expression of survivin. B- To confirm the inhibition of AKT and PI3K, HER2 induced MCF7 cells were incubated for 48 hours with specific inhibitors of PI3K (10 μM LY294002) AKT1/2/3 (15 μM AKT IV Inhibitor). ERK1/2 (10 μM U0126) and without inhibitor MCF-7 cells were used as controls, and analyzed by western blotting for expression of phospho-ribosomal protein S6 (S235).
Figure Legend Snippet: Signaling pathways involved in survivin regulation in HER2 over expressing MCF7cells . A- HER2 induced MCF7 cells were incubated for 48 hours with specific inhibitors of PI3K (10 μM LY294002) AKT1/2/3 (15 μM AKT IV Inhibitor) and ERK1/2 (10 μM U0126) or without inhibitor (control) and analyzed by western blotting for expression of survivin. B- To confirm the inhibition of AKT and PI3K, HER2 induced MCF7 cells were incubated for 48 hours with specific inhibitors of PI3K (10 μM LY294002) AKT1/2/3 (15 μM AKT IV Inhibitor). ERK1/2 (10 μM U0126) and without inhibitor MCF-7 cells were used as controls, and analyzed by western blotting for expression of phospho-ribosomal protein S6 (S235).

Techniques Used: Expressing, Incubation, Western Blot, Inhibition

5) Product Images from "Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways"

Article Title: Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0133699

A model depicting the mechanisms of p17 modulating Tpr, p53, AKT, p21, PTEN, mTORC1 that govern cell cycle and autophagosome formation. This study establishes a new regulatory network of p17 linking Tpr, p53, p21, PTEN, mTORC1, and Rb. p17 suppresses Tpr leading to p53 and p21 nuclear accumulation, which in turn activates p53, p21, and PTEN. Furthermore, it also serves as a positive regulator of PTEN. Activation of PTEN leads to inhibition of ERK and AKT that result in mTORC1 inhibition as well as cyclin D1 and CDK 4 inhibition, leading to Rb activation. This study provides evidences demonstrating that p17 regulates cell cycle through Tpr/p53/PTEN/AKT and Tpr/p53/p21 signaling pathways. By suppressing Tpr, p17 is able to negatively regulate PI3K/AKT/mTORC1 and consequently induce cellular translation shutoff and autophagosome formation enhancing virus replication. →: activation;⊥: Inhibition.
Figure Legend Snippet: A model depicting the mechanisms of p17 modulating Tpr, p53, AKT, p21, PTEN, mTORC1 that govern cell cycle and autophagosome formation. This study establishes a new regulatory network of p17 linking Tpr, p53, p21, PTEN, mTORC1, and Rb. p17 suppresses Tpr leading to p53 and p21 nuclear accumulation, which in turn activates p53, p21, and PTEN. Furthermore, it also serves as a positive regulator of PTEN. Activation of PTEN leads to inhibition of ERK and AKT that result in mTORC1 inhibition as well as cyclin D1 and CDK 4 inhibition, leading to Rb activation. This study provides evidences demonstrating that p17 regulates cell cycle through Tpr/p53/PTEN/AKT and Tpr/p53/p21 signaling pathways. By suppressing Tpr, p17 is able to negatively regulate PI3K/AKT/mTORC1 and consequently induce cellular translation shutoff and autophagosome formation enhancing virus replication. →: activation;⊥: Inhibition.

Techniques Used: Activation Assay, Inhibition

p17 negatively regulates PI3K/AKT/mTOR signaling pathway. (A-B) Vero cells were transfected with pcDNA3.1-p17 and pcDNA3.1 (vector only) plasmid, respectively for 24 hours. Whole cell lysates were collected at the indicated time points, and the levels of PI3K and its downstream molecules were examined by Western blot assay with the indicated antibodies. (B)Vero and DF-1 cells were co-transfected with both pcDNA3.1-p17 and p53 shRNAs for 24 hours, followed by Western blot analysis with indicated antibodies. Cells were also co-transfected with pCDNA3.1- p17 and respective negative controls (scrambled shRNAs and pGFP-V-RS vector) for 24 hours. (C) To study whether the negative control p17 mutant (1–118) can affect the levels of p-PTEN, p-AKT, p-mTOR, and LC3-II, vero cells were transfected with the p17 mutant (1–118) plasmid for 24 hours. Similar results were obtained from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins at 0 h or mock were considered 1-fold.
Figure Legend Snippet: p17 negatively regulates PI3K/AKT/mTOR signaling pathway. (A-B) Vero cells were transfected with pcDNA3.1-p17 and pcDNA3.1 (vector only) plasmid, respectively for 24 hours. Whole cell lysates were collected at the indicated time points, and the levels of PI3K and its downstream molecules were examined by Western blot assay with the indicated antibodies. (B)Vero and DF-1 cells were co-transfected with both pcDNA3.1-p17 and p53 shRNAs for 24 hours, followed by Western blot analysis with indicated antibodies. Cells were also co-transfected with pCDNA3.1- p17 and respective negative controls (scrambled shRNAs and pGFP-V-RS vector) for 24 hours. (C) To study whether the negative control p17 mutant (1–118) can affect the levels of p-PTEN, p-AKT, p-mTOR, and LC3-II, vero cells were transfected with the p17 mutant (1–118) plasmid for 24 hours. Similar results were obtained from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins at 0 h or mock were considered 1-fold.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Negative Control, Mutagenesis, Activation Assay

6) Product Images from "Increased Interleukin-23 in Hashimoto’s Thyroiditis Disease Induces Autophagy Suppression and Reactive Oxygen Species Accumulation"

Article Title: Increased Interleukin-23 in Hashimoto’s Thyroiditis Disease Induces Autophagy Suppression and Reactive Oxygen Species Accumulation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00096

Interleukin-23 (IL-23) induces AKT/mTOR/NF-κB signaling pathway activation. Nthy-ori 3-1 cells were treated with IL-23 (50 ng/mL) at different time points, and changes of AKT, p-AKT (Ser473), mTOR, p-mTOR (Ser2448), S6k, p-S6k (Thr389), S6, and p-S6 (Ser235/236) expression levels (A) , as well as NF-κB p65, p-NF-κB p65 (Ser536), Stat3, and p-Stat3 (Ser727) expression levels (B) , were determined by western blot. (C) Nthy-ori 3-1 cells were treated with IL-23 (50 ng/mL) and/or anti-IL-23 pre-treatment (5 µg/mL), changes of AKT, p-AKT (Ser473), S6k, p-S6k (Thr389), NF-κB p65, p-NF-κB p65 (Ser536), Stat3, and p-Stat3 (Ser727) expression levels were analyzed by western blot. (D) The change of LC3B-II expression in Nthy-ori 3-1 cells was analyzed at 3 h by western blot in the presence of IL-23 (50 ng/mL) with or without Stattic (10 µM) treatment. The results shown are representative of three replicates.
Figure Legend Snippet: Interleukin-23 (IL-23) induces AKT/mTOR/NF-κB signaling pathway activation. Nthy-ori 3-1 cells were treated with IL-23 (50 ng/mL) at different time points, and changes of AKT, p-AKT (Ser473), mTOR, p-mTOR (Ser2448), S6k, p-S6k (Thr389), S6, and p-S6 (Ser235/236) expression levels (A) , as well as NF-κB p65, p-NF-κB p65 (Ser536), Stat3, and p-Stat3 (Ser727) expression levels (B) , were determined by western blot. (C) Nthy-ori 3-1 cells were treated with IL-23 (50 ng/mL) and/or anti-IL-23 pre-treatment (5 µg/mL), changes of AKT, p-AKT (Ser473), S6k, p-S6k (Thr389), NF-κB p65, p-NF-κB p65 (Ser536), Stat3, and p-Stat3 (Ser727) expression levels were analyzed by western blot. (D) The change of LC3B-II expression in Nthy-ori 3-1 cells was analyzed at 3 h by western blot in the presence of IL-23 (50 ng/mL) with or without Stattic (10 µM) treatment. The results shown are representative of three replicates.

Techniques Used: Activation Assay, Expressing, Western Blot

AKT, p-AKT, mTOR, p-mTOR, S6, p-S6, NF-κB p65, and p-NF-κB p65 expression levels in Hashimoto’s thyroiditis (HT) patient thyroid glands. Representative results from HT patients ( n = 10) and control tissues ( n = 5) by immunohistochemistry (IHC) staining for AKT and p-AKT (A) , mTOR and p-mTOR (B) , S6 and p-S6 (C) , and NF-κB and p-NF-κB (D) are shown (scale bars, 50 µm). The brown regions represent positive expression. The slides were analyzed under a 400× microscope equipped with a camera. The IHC quantification results from all samples are shown in the right panel. Significant differences and P values are calculated with the Mann–Whitney U test. ns, not significant. *** P
Figure Legend Snippet: AKT, p-AKT, mTOR, p-mTOR, S6, p-S6, NF-κB p65, and p-NF-κB p65 expression levels in Hashimoto’s thyroiditis (HT) patient thyroid glands. Representative results from HT patients ( n = 10) and control tissues ( n = 5) by immunohistochemistry (IHC) staining for AKT and p-AKT (A) , mTOR and p-mTOR (B) , S6 and p-S6 (C) , and NF-κB and p-NF-κB (D) are shown (scale bars, 50 µm). The brown regions represent positive expression. The slides were analyzed under a 400× microscope equipped with a camera. The IHC quantification results from all samples are shown in the right panel. Significant differences and P values are calculated with the Mann–Whitney U test. ns, not significant. *** P

Techniques Used: Expressing, Immunohistochemistry, Staining, Microscopy, MANN-WHITNEY

7) Product Images from "Trail (TNF-related apoptosis-inducing ligand) induces an inflammatory response in human adipocytes"

Article Title: Trail (TNF-related apoptosis-inducing ligand) induces an inflammatory response in human adipocytes

Journal: Scientific Reports

doi: 10.1038/s41598-017-05932-7

TRAIL induces the phosphorylation of ERK1/2. ( A ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). The phosphorylation of ERK1/2, JNK and AKT was determined by Western blot. ( B – F ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the MEK1/2 inhibitor PD-0325901 (100 nM). After 6 hours, the phosphorylation of ERK1/2 and IκBα was analyzed by Western blot ( B ). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, IL-6 ( C ), IL-8 ( D ), MCP-1 ( E ) and CCL-20 ( F ) was analyzed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p
Figure Legend Snippet: TRAIL induces the phosphorylation of ERK1/2. ( A ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). The phosphorylation of ERK1/2, JNK and AKT was determined by Western blot. ( B – F ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the MEK1/2 inhibitor PD-0325901 (100 nM). After 6 hours, the phosphorylation of ERK1/2 and IκBα was analyzed by Western blot ( B ). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, IL-6 ( C ), IL-8 ( D ), MCP-1 ( E ) and CCL-20 ( F ) was analyzed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p

Techniques Used: Isolation, Western Blot, Real-time Polymerase Chain Reaction

8) Product Images from "Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy"

Article Title: Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy

Journal: Skeletal Muscle

doi: 10.1186/s13395-016-0091-9

mTOR is activated in aged, fasted Myf5/ Fktn KO muscle. a Western blot analysis of solubilized protein from the hind limb muscle of Myf5/ Fktn LC and KO mice. b Quantification of Akt phosphorylation at T308 and S473, mTOR phosphorylation at S2448, S6 phosphorylation at S235/236, relative to total Akt, mTOR, and S6 protein, respectively, as a measure of protein activation. mTOR phosphorylation at S2448 normalized to total mTOR is significantly increased in KO muscle. Two-tailed Student’s t test; * p
Figure Legend Snippet: mTOR is activated in aged, fasted Myf5/ Fktn KO muscle. a Western blot analysis of solubilized protein from the hind limb muscle of Myf5/ Fktn LC and KO mice. b Quantification of Akt phosphorylation at T308 and S473, mTOR phosphorylation at S2448, S6 phosphorylation at S235/236, relative to total Akt, mTOR, and S6 protein, respectively, as a measure of protein activation. mTOR phosphorylation at S2448 normalized to total mTOR is significantly increased in KO muscle. Two-tailed Student’s t test; * p

Techniques Used: Western Blot, Mouse Assay, Activation Assay, Two Tailed Test

Akt/mTOR signaling is unchanged following loss of αDG glycosylation. a Representative images of littermate (Tam LC) or inducible knockout (Tam iKO) iliopsoas. H E and αDG glyco images are shown. Scale bar = 100 μm. b Western blot analysis of solubilized skeletal muscle from hind limbs of Tam LC or Tam iKO mice. c Quantification of Akt phosphorylation at T308 and S473, mTOR phosphorylation at S2448, and S6 phosphorylation at S235/236 relative to total Akt, mTOR, and S6 protein, respectively, as a measure of protein activation. Data are presented as mean ± SEM. n = 9 per group
Figure Legend Snippet: Akt/mTOR signaling is unchanged following loss of αDG glycosylation. a Representative images of littermate (Tam LC) or inducible knockout (Tam iKO) iliopsoas. H E and αDG glyco images are shown. Scale bar = 100 μm. b Western blot analysis of solubilized skeletal muscle from hind limbs of Tam LC or Tam iKO mice. c Quantification of Akt phosphorylation at T308 and S473, mTOR phosphorylation at S2448, and S6 phosphorylation at S235/236 relative to total Akt, mTOR, and S6 protein, respectively, as a measure of protein activation. Data are presented as mean ± SEM. n = 9 per group

Techniques Used: Knock-Out, Western Blot, Mouse Assay, Activation Assay

9) Product Images from "Luteolin Induces Apoptosis and Autophagy in Mouse Macrophage ANA-1 Cells via the Bcl-2 Pathway"

Article Title: Luteolin Induces Apoptosis and Autophagy in Mouse Macrophage ANA-1 Cells via the Bcl-2 Pathway

Journal: Journal of Immunology Research

doi: 10.1155/2018/4623919

The effects of luteolin on ERK/p38/Akt in the ANA-1 cells. (a) Representative Western blot results showing phospho-ERK1/2/ERK1/2, phospho-p38/p38, and phospho-Akt/Akt expression in the ANA-1 cells. (b) The relative expression of proteins compared with the control. ∗ p
Figure Legend Snippet: The effects of luteolin on ERK/p38/Akt in the ANA-1 cells. (a) Representative Western blot results showing phospho-ERK1/2/ERK1/2, phospho-p38/p38, and phospho-Akt/Akt expression in the ANA-1 cells. (b) The relative expression of proteins compared with the control. ∗ p

Techniques Used: Western Blot, Expressing

10) Product Images from "Ursolic Acid Attenuates High Glucose-Mediated Mesangial Cell Injury by Inhibiting the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) Signaling Pathway"

Article Title: Ursolic Acid Attenuates High Glucose-Mediated Mesangial Cell Injury by Inhibiting the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin (PI3K/Akt/mTOR) Signaling Pathway

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.907814

( A–C ) Western blotting was used to detect PI3K/Akt/mTOR pathway activity in mesangial cells of each group. NG: normal glucose group (5 mmol/L glucose); Mannitol: hypertonic control group (5 mmol/L glucose+24.5 mmol/L mannitol); HG: high glucose group (30 mmol/L glucose); HG+UA: ursolic acid treatment group (30 mmol/L glucose+1.0 mmol/L ursolic acid). # p
Figure Legend Snippet: ( A–C ) Western blotting was used to detect PI3K/Akt/mTOR pathway activity in mesangial cells of each group. NG: normal glucose group (5 mmol/L glucose); Mannitol: hypertonic control group (5 mmol/L glucose+24.5 mmol/L mannitol); HG: high glucose group (30 mmol/L glucose); HG+UA: ursolic acid treatment group (30 mmol/L glucose+1.0 mmol/L ursolic acid). # p

Techniques Used: Western Blot, Activity Assay

11) Product Images from "USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells"

Article Title: USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-018-0291-5

( a ) HCC827 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed by immuno-blotting using anti-pERK1/2, anti-pAkt, anti-pSrc and, anti-pEGFR (Tyr1068). Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, utilising Erk1/2, Akt, Src, EGFR and anti-tubulin antibodies. ( b ) H1975 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed as before. Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, as before
Figure Legend Snippet: ( a ) HCC827 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed by immuno-blotting using anti-pERK1/2, anti-pAkt, anti-pSrc and, anti-pEGFR (Tyr1068). Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, utilising Erk1/2, Akt, Src, EGFR and anti-tubulin antibodies. ( b ) H1975 cells were transfected as indicated. 72 h post transfection the cells were starved in serum free medium for 3 h. Whole cell lysates were harvested and levels of phosphorylated Erk1/2, Akt, Src and EGFR were assessed as before. Total protein levels of Erk, Akt, Src and EGFR were also assessed in addition to α-tubulin, as before

Techniques Used: Transfection

12) Product Images from "Characterization of AQX-1125, a small-molecule SHIP1 activator"

Article Title: Characterization of AQX-1125, a small-molecule SHIP1 activator

Journal: British Journal of Pharmacology

doi: 10.1111/bph.12039

AQX-1125 inhibits the phosphorylation of Akt (S473) in MOLT-4 but not Jurkat cells. The effect of AQX-1125 on the level of pAkt (S473) in serum starved and IGF-1 stimulated MOLT-4 and Jurkat cells were examined. Data show representative results of three
Figure Legend Snippet: AQX-1125 inhibits the phosphorylation of Akt (S473) in MOLT-4 but not Jurkat cells. The effect of AQX-1125 on the level of pAkt (S473) in serum starved and IGF-1 stimulated MOLT-4 and Jurkat cells were examined. Data show representative results of three

Techniques Used:

13) Product Images from "Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6"

Article Title: Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6

Journal: Scientific Reports

doi: 10.1038/s41598-017-05510-x

p17 interferes with the formation of the CDK2/cyclin A2 complex, which impedes Akt phosphorylation. ( A ) Levels of CDK2, cyclin A2, p-Akt (S473), p-GSK3α (S21), p-GSK3β (S9), and p-Rb (S249) in ARV-infected and p17-transfected Vero cells were examined. Cells were collected at the indicated points, and whole cell lysates were harvested for Western blot assays. p17 (1–118)-transfected and mock-infected cells were used as negative controls. β-actin was included as a loading control. ( B ) The level of CDK2 was examined in Vero cells without treatment or pretreated with MG132 followed by mock infection, ARV infection, and p17 transfection, respectively. Levels of CDK 2 mRNA in ARV-infected and pcDNA3.1-flag-p17-transfected Vero cells were analyzed by semi-quantitative RT-PCR. Mock infection (cells alone) was used as a negative control. The graph represents the mean ± SD calculated from three independent experiments. ( C ) The amount of CDK2 and cyclin A2 association were examined in either ARV-infected or p17-transfected Vero cells. ( D ) An in vitro GST pull-down assay was carried out. Elution fractions were boiled and examined by Western blot analysis. 30% total input of TrxA-His-17 or TrxA-His-17(1–118) mutant represented the internal loading control. ( E ) To confirm whether CDK2 phosphorylates Akt, knockdown of CDK2 with an shRNA and overexpression of CDK2 in p17-transfected cells were carried out, followed by Western blot analysis with indicated antibodies. For negative controls, cells were transfected as indicated. ( F ) To test whether insulin and CDK2 overexpression counteract the inhibitory effect of p17 on mTORC2 complex association, Vero cells were pretreated with insulin (0.2 μm) or transfected with pCI-neo-CDK2 plasmid for 3 hours, respectively, followed by transfection with pcDNA3.1-Flag-p17 for 18 hours. Vero cells were collected and washed twice in phosphate-buffered saline (PBS) and scraped in 200 μl of CHAPS lysis buffer. ( G ) To determine the effects of Akt and CDK2 on ARV replication, individual 24-well plates of Vero cells were infected with ARV at an MOI of 5 for 6 hours, followed by transfection with Akt and CDK2 shRNAs or the pCI-neo-CDK2 plasmid for 24 hours, respectively. The ARV-infected cell supernatant was collected at 24 hpi for determining virus titer. All the data shown represent the mean ± SD calculated from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The uncropped blots with molecular weights are shown in Figs S7 and S8 .
Figure Legend Snippet: p17 interferes with the formation of the CDK2/cyclin A2 complex, which impedes Akt phosphorylation. ( A ) Levels of CDK2, cyclin A2, p-Akt (S473), p-GSK3α (S21), p-GSK3β (S9), and p-Rb (S249) in ARV-infected and p17-transfected Vero cells were examined. Cells were collected at the indicated points, and whole cell lysates were harvested for Western blot assays. p17 (1–118)-transfected and mock-infected cells were used as negative controls. β-actin was included as a loading control. ( B ) The level of CDK2 was examined in Vero cells without treatment or pretreated with MG132 followed by mock infection, ARV infection, and p17 transfection, respectively. Levels of CDK 2 mRNA in ARV-infected and pcDNA3.1-flag-p17-transfected Vero cells were analyzed by semi-quantitative RT-PCR. Mock infection (cells alone) was used as a negative control. The graph represents the mean ± SD calculated from three independent experiments. ( C ) The amount of CDK2 and cyclin A2 association were examined in either ARV-infected or p17-transfected Vero cells. ( D ) An in vitro GST pull-down assay was carried out. Elution fractions were boiled and examined by Western blot analysis. 30% total input of TrxA-His-17 or TrxA-His-17(1–118) mutant represented the internal loading control. ( E ) To confirm whether CDK2 phosphorylates Akt, knockdown of CDK2 with an shRNA and overexpression of CDK2 in p17-transfected cells were carried out, followed by Western blot analysis with indicated antibodies. For negative controls, cells were transfected as indicated. ( F ) To test whether insulin and CDK2 overexpression counteract the inhibitory effect of p17 on mTORC2 complex association, Vero cells were pretreated with insulin (0.2 μm) or transfected with pCI-neo-CDK2 plasmid for 3 hours, respectively, followed by transfection with pcDNA3.1-Flag-p17 for 18 hours. Vero cells were collected and washed twice in phosphate-buffered saline (PBS) and scraped in 200 μl of CHAPS lysis buffer. ( G ) To determine the effects of Akt and CDK2 on ARV replication, individual 24-well plates of Vero cells were infected with ARV at an MOI of 5 for 6 hours, followed by transfection with Akt and CDK2 shRNAs or the pCI-neo-CDK2 plasmid for 24 hours, respectively. The ARV-infected cell supernatant was collected at 24 hpi for determining virus titer. All the data shown represent the mean ± SD calculated from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The uncropped blots with molecular weights are shown in Figs S7 and S8 .

Techniques Used: Infection, Transfection, Western Blot, Quantitative RT-PCR, Negative Control, In Vitro, Pull Down Assay, Mutagenesis, shRNA, Over Expression, Plasmid Preparation, Lysis, Activation Assay

ARV σA protein enhances the proteasome activity and expression level of PSMB6. ( A ) Vero cell lysates from ARV-infected, σA-transfected, and PSMB6- depleted cells with ARV at an MOI of 10 for 24 hours were used to quantify relative proteasome activity. ( B ) Examination of the PSMB6 levels. Vero cells were infected with ARV at an MOI of 10 at indicated time points, followed by Western blot assay with an anti-PSMB6 antibody. ( C ) Analysis of mRNA levels of PSMB6 and other subunits by real-time RT-PCR.The data reveal that PSMB6 is transcriptionally upregulated by σA. ( D ) To confirm whether PSMB6 mediates ribosomal protein ubiquitin-proteasome-mediated degradation and inhibits Akt phosphorylation at S473, Vero cells were transfected with plasmids overexpressing PSMB6 followed by Western blot assay with the indicated antibodies ( E ) In the presence of MG132, the decrease in the levels of p-Akt (S473) in ARV-infected vero cells could be reversed in ARV-infected cells. ( F ) The levels of PSMB6, Rpl26, Rpl27, and p-Akt (S473) were examined in ARV-infected cells co-transfected with an shRNA against σA. ( G ) To confirm whether both PSMB6 and MDM2 mediate ribosomal proteins degradation, knockdown of either PSMB6 or MDM2 with shRNAs was performed followed by Western blot analysis with the indicated antibodies. ( H ) Vero cells were transfected with pcDNA3.1-p17 or co-transfected with pcDNA3.1-p17 and pcDNA3.1-σA plasmids for 24 hours, respectively, followed by Western blot assays with the indicated antibodies. ( I ) Individual 24-well plates of Vero cells were transfected with a PSMB6 shRNA for 24 hours, followed by ARV infection at an MOI of 5 for 24 hours. The ARV-infected cell supernatant was collected at 24 hpi for determining the virus titer. All data shown represent the mean ± SD calculated from three independent experiments. ( J ) A model depicting the cooperation between p17 and σA proteins of ARV to trigger ribosomal protein degradation is shown. The protein levels were normalized to those for β-actin. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The activation and inactivation folds indicated below each lane were normalized against against values for the mock control or at 0 h. The uncropped blots with molecular weights are shown in Figs S5 and S6 .
Figure Legend Snippet: ARV σA protein enhances the proteasome activity and expression level of PSMB6. ( A ) Vero cell lysates from ARV-infected, σA-transfected, and PSMB6- depleted cells with ARV at an MOI of 10 for 24 hours were used to quantify relative proteasome activity. ( B ) Examination of the PSMB6 levels. Vero cells were infected with ARV at an MOI of 10 at indicated time points, followed by Western blot assay with an anti-PSMB6 antibody. ( C ) Analysis of mRNA levels of PSMB6 and other subunits by real-time RT-PCR.The data reveal that PSMB6 is transcriptionally upregulated by σA. ( D ) To confirm whether PSMB6 mediates ribosomal protein ubiquitin-proteasome-mediated degradation and inhibits Akt phosphorylation at S473, Vero cells were transfected with plasmids overexpressing PSMB6 followed by Western blot assay with the indicated antibodies ( E ) In the presence of MG132, the decrease in the levels of p-Akt (S473) in ARV-infected vero cells could be reversed in ARV-infected cells. ( F ) The levels of PSMB6, Rpl26, Rpl27, and p-Akt (S473) were examined in ARV-infected cells co-transfected with an shRNA against σA. ( G ) To confirm whether both PSMB6 and MDM2 mediate ribosomal proteins degradation, knockdown of either PSMB6 or MDM2 with shRNAs was performed followed by Western blot analysis with the indicated antibodies. ( H ) Vero cells were transfected with pcDNA3.1-p17 or co-transfected with pcDNA3.1-p17 and pcDNA3.1-σA plasmids for 24 hours, respectively, followed by Western blot assays with the indicated antibodies. ( I ) Individual 24-well plates of Vero cells were transfected with a PSMB6 shRNA for 24 hours, followed by ARV infection at an MOI of 5 for 24 hours. The ARV-infected cell supernatant was collected at 24 hpi for determining the virus titer. All data shown represent the mean ± SD calculated from three independent experiments. ( J ) A model depicting the cooperation between p17 and σA proteins of ARV to trigger ribosomal protein degradation is shown. The protein levels were normalized to those for β-actin. The levels of indicated proteins in the mock control or at 0 h were considered 1-fold. The activation and inactivation folds indicated below each lane were normalized against against values for the mock control or at 0 h. The uncropped blots with molecular weights are shown in Figs S5 and S6 .

Techniques Used: Activity Assay, Expressing, Infection, Transfection, Western Blot, Quantitative RT-PCR, shRNA, Activation Assay

A model depicting the mechanism of avian reovirus p17 protein cooperating with σA to regulate autophagy and the cell cycle via suppression of mTOR and CDK2/cyclinA2 complexes and activation of proteasome subunit PSMB6 to facilitate ribosomal protein degradation. p17-mediated reduction of ribosomal proteins occurs via the E3 ligase MDM2 to mediate ribosomal protein polyubiquitylation. We also uncovered that ARV σA protein increases the proteasome activity and expression level of proteasome subunit PSMB6. Furthermore, p17 binds to and inhibits the CDK2/cyclin A2 complex, further reducing phosphorylation of Akt S473. Both p17 and σA proteins act cooperatively to inhibit mTORC2, which inhibits Akt and activates Beclin, inducing autophagy. The present study provides mechanistic insights into cooperation between p17 and σA proteins of ARV to negatively regulate Akt by downregulation of mTORC2 and CDK2/cyclin A2 complexes and upregulation of proteasome subunit PSMB6, which together induces autophagy.
Figure Legend Snippet: A model depicting the mechanism of avian reovirus p17 protein cooperating with σA to regulate autophagy and the cell cycle via suppression of mTOR and CDK2/cyclinA2 complexes and activation of proteasome subunit PSMB6 to facilitate ribosomal protein degradation. p17-mediated reduction of ribosomal proteins occurs via the E3 ligase MDM2 to mediate ribosomal protein polyubiquitylation. We also uncovered that ARV σA protein increases the proteasome activity and expression level of proteasome subunit PSMB6. Furthermore, p17 binds to and inhibits the CDK2/cyclin A2 complex, further reducing phosphorylation of Akt S473. Both p17 and σA proteins act cooperatively to inhibit mTORC2, which inhibits Akt and activates Beclin, inducing autophagy. The present study provides mechanistic insights into cooperation between p17 and σA proteins of ARV to negatively regulate Akt by downregulation of mTORC2 and CDK2/cyclin A2 complexes and upregulation of proteasome subunit PSMB6, which together induces autophagy.

Techniques Used: Activation Assay, Activity Assay, Expressing, Activated Clotting Time Assay

p17 deregulates mTORC2 assembly. ( A ) To study the effect of insulin on ribosomal proteins and Akt phosphorylation at S473, cells were pretreated with insulin (0.2 μm) for 1 hour, followed by transfection with pcDNA3.1-Flag-p17 for 24 hours at 37 °C. Whole cell lysates were harvested for Western blot assays with the indicated antibodies. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against values for the mock control. those at mock. The levels of indicated proteins in the mock control were considered 1-fold. ( B ) Upper panel: in co-immunoprecipitation experiments, the binding of rictor, mSN1, and MlST8 to mTOR was examined in ARV-infected or p17-transfected Vero cells. Cells were mock-infected or infected with ARV at an MOI of 10 and transfected with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag (vector only) plasmid for 24 hours. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot analysis, and proteins were detected with the indicated antibodies. Lower panel: Data were obtained in three independent experiments, error bars indicate the means ± SD. ( C ) Upper panel: for analysis of the effect of insulin on mTORC2, cells were treated with insulin (0.2 uM) for 1 hour, followed by transfection of cells with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag plasmid for 24 hours at 37 °C. The interaction of mTOR and rictor with Rpl7 and Rpl26 was examined by co-immunoprecipitation experiments described in panel A. Lower panel: Data shown represent the mean ± SD calculated from three independent experiments. ( D ) To study whether MDM2 and PSMB6 affect the association of mTORC2 and ribosomal proteins, depletion of MDM2 and PSMB6 with shRNAs was performed. The uncropped blots with molecular weights are shown in Figs S6 and S7 .
Figure Legend Snippet: p17 deregulates mTORC2 assembly. ( A ) To study the effect of insulin on ribosomal proteins and Akt phosphorylation at S473, cells were pretreated with insulin (0.2 μm) for 1 hour, followed by transfection with pcDNA3.1-Flag-p17 for 24 hours at 37 °C. Whole cell lysates were harvested for Western blot assays with the indicated antibodies. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against values for the mock control. those at mock. The levels of indicated proteins in the mock control were considered 1-fold. ( B ) Upper panel: in co-immunoprecipitation experiments, the binding of rictor, mSN1, and MlST8 to mTOR was examined in ARV-infected or p17-transfected Vero cells. Cells were mock-infected or infected with ARV at an MOI of 10 and transfected with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag (vector only) plasmid for 24 hours. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot analysis, and proteins were detected with the indicated antibodies. Lower panel: Data were obtained in three independent experiments, error bars indicate the means ± SD. ( C ) Upper panel: for analysis of the effect of insulin on mTORC2, cells were treated with insulin (0.2 uM) for 1 hour, followed by transfection of cells with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag plasmid for 24 hours at 37 °C. The interaction of mTOR and rictor with Rpl7 and Rpl26 was examined by co-immunoprecipitation experiments described in panel A. Lower panel: Data shown represent the mean ± SD calculated from three independent experiments. ( D ) To study whether MDM2 and PSMB6 affect the association of mTORC2 and ribosomal proteins, depletion of MDM2 and PSMB6 with shRNAs was performed. The uncropped blots with molecular weights are shown in Figs S6 and S7 .

Techniques Used: Transfection, Western Blot, Activation Assay, Immunoprecipitation, Binding Assay, Infection, Plasmid Preparation, SDS Page

p17 inactivates Akt reducing the association of Beclin 1 and 14-3-3. Vero cells were infected with ARV at an MOI of 10 ( A ) or ( B ) transfected with pcDNA3.1-Flag-p17 or pcDNA3.1-Flag-σA plasmids for 24 hours and protein analyzed by Western blot assays. ( C ) In co-immunoprecipitation experiments, the amounts of 14-3-3-Beclin 1 and 14-3-3-vimentin association were examined in p17-transfected Vero cells. Western blot assays of Beclin 1, 14-3-3 and vimentin contained in 14-3-3 immunoprecipitates were performed. Rabbit IgG was used as a negative control. ( D,E ) In reciprocal co-immunoprecipitation experiments, the binding of 14-3-3 and Beclin 1 was examined in p17-transfected and PTEN shRNA-cotransfected Vero cells ( D ) as well as CDK2 overexpression (pCI-neo-CDK2 vector) in p17-transfected Vero cells ( E ). ( F ) To study whether p17-mediated Akt inhibition leads to an increased amount of Beclin 1 and 14-3-3 association, the amounts of Beclin 1 and 14-3-3 association in Akt-knockdown Vero cells or in p17-transfected or mock-transfected cells were examined. ( G ) In reciprocal co-immunoprecipitation experiments, the binding of 14-3-3 and Beclin 1 was examined in either p17-transfected or PSMB6 shRNA-cotransfected Vero cells. Western blot analysis of 14-3-3 and Beclin 1 contained in 14-3-3 or Beclin 1 immunoprecipates was performed. ( H ) In reciprocal co-immunoprecipitation experiments, the binding of 14-3-3 and Beclin 1 was examined in either ARV-infected or σA shRNA-transfected Vero cells. Western blot analysis of 14-3-3 and Beclin 1 contained in 14-3-3 or Beclin 1 immunoprecipitates was performed. ( I ) Data shown represent the mean ± SD calculated from three independent experiments in distinct shRNA treatments as shown in Fig. 5C–H. The amounts of Beclin1/14-3-3 association were normalized against the value in the negative control. The level of the negative control was considered 1-fold. Data shown represent the mean ± SD calculated from three independent experiments. Controls of knockdown of PTEN, Akt, CDK2, PSMB6, and σA in Fig. 5D,E,F,G,H are shown. The uncropped blots of blots with molecular weights are shown in Figs S8 and S9 .
Figure Legend Snippet: p17 inactivates Akt reducing the association of Beclin 1 and 14-3-3. Vero cells were infected with ARV at an MOI of 10 ( A ) or ( B ) transfected with pcDNA3.1-Flag-p17 or pcDNA3.1-Flag-σA plasmids for 24 hours and protein analyzed by Western blot assays. ( C ) In co-immunoprecipitation experiments, the amounts of 14-3-3-Beclin 1 and 14-3-3-vimentin association were examined in p17-transfected Vero cells. Western blot assays of Beclin 1, 14-3-3 and vimentin contained in 14-3-3 immunoprecipitates were performed. Rabbit IgG was used as a negative control. ( D,E ) In reciprocal co-immunoprecipitation experiments, the binding of 14-3-3 and Beclin 1 was examined in p17-transfected and PTEN shRNA-cotransfected Vero cells ( D ) as well as CDK2 overexpression (pCI-neo-CDK2 vector) in p17-transfected Vero cells ( E ). ( F ) To study whether p17-mediated Akt inhibition leads to an increased amount of Beclin 1 and 14-3-3 association, the amounts of Beclin 1 and 14-3-3 association in Akt-knockdown Vero cells or in p17-transfected or mock-transfected cells were examined. ( G ) In reciprocal co-immunoprecipitation experiments, the binding of 14-3-3 and Beclin 1 was examined in either p17-transfected or PSMB6 shRNA-cotransfected Vero cells. Western blot analysis of 14-3-3 and Beclin 1 contained in 14-3-3 or Beclin 1 immunoprecipates was performed. ( H ) In reciprocal co-immunoprecipitation experiments, the binding of 14-3-3 and Beclin 1 was examined in either ARV-infected or σA shRNA-transfected Vero cells. Western blot analysis of 14-3-3 and Beclin 1 contained in 14-3-3 or Beclin 1 immunoprecipitates was performed. ( I ) Data shown represent the mean ± SD calculated from three independent experiments in distinct shRNA treatments as shown in Fig. 5C–H. The amounts of Beclin1/14-3-3 association were normalized against the value in the negative control. The level of the negative control was considered 1-fold. Data shown represent the mean ± SD calculated from three independent experiments. Controls of knockdown of PTEN, Akt, CDK2, PSMB6, and σA in Fig. 5D,E,F,G,H are shown. The uncropped blots of blots with molecular weights are shown in Figs S8 and S9 .

Techniques Used: Infection, Transfection, Western Blot, Immunoprecipitation, Negative Control, Binding Assay, shRNA, Over Expression, Plasmid Preparation, Inhibition

A model depicting the mechanism of avian reovirus p17 protein cooperating with σA to regulate autophagy and the cell cycle via suppression of mTOR and CDK2/cyclinA2 complexes and activation of proteasome subunit PSMB6 to facilitate ribosomal protein degradation. p17-mediated reduction of ribosomal proteins occurs via the E3 ligase MDM2 to mediate ribosomal protein polyubiquitylation. We also uncovered that ARV σA protein increases the proteasome activity and expression level of proteasome subunit PSMB6. Furthermore, p17 binds to and inhibits the CDK2/cyclin A2 complex, further reducing phosphorylation of Akt S473. Both p17 and σA proteins act cooperatively to inhibit mTORC2, which inhibits Akt and activates Beclin, inducing autophagy. The present study provides mechanistic insights into cooperation between p17 and σA proteins of ARV to negatively regulate Akt by downregulation of mTORC2 and CDK2/cyclin A2 complexes and upregulation of proteasome subunit PSMB6, which together induces autophagy.
Figure Legend Snippet: A model depicting the mechanism of avian reovirus p17 protein cooperating with σA to regulate autophagy and the cell cycle via suppression of mTOR and CDK2/cyclinA2 complexes and activation of proteasome subunit PSMB6 to facilitate ribosomal protein degradation. p17-mediated reduction of ribosomal proteins occurs via the E3 ligase MDM2 to mediate ribosomal protein polyubiquitylation. We also uncovered that ARV σA protein increases the proteasome activity and expression level of proteasome subunit PSMB6. Furthermore, p17 binds to and inhibits the CDK2/cyclin A2 complex, further reducing phosphorylation of Akt S473. Both p17 and σA proteins act cooperatively to inhibit mTORC2, which inhibits Akt and activates Beclin, inducing autophagy. The present study provides mechanistic insights into cooperation between p17 and σA proteins of ARV to negatively regulate Akt by downregulation of mTORC2 and CDK2/cyclin A2 complexes and upregulation of proteasome subunit PSMB6, which together induces autophagy.

Techniques Used: Activation Assay, Activity Assay, Expressing, Activated Clotting Time Assay

p17 deregulates mTORC2 assembly. ( A ) To study the effect of insulin on ribosomal proteins and Akt phosphorylation at S473, cells were pretreated with insulin (0.2 μm) for 1 hour, followed by transfection with pcDNA3.1-Flag-p17 for 24 hours at 37 °C. Whole cell lysates were harvested for Western blot assays with the indicated antibodies. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against values for the mock control. those at mock. The levels of indicated proteins in the mock control were considered 1-fold. ( B ) Upper panel: in co-immunoprecipitation experiments, the binding of rictor, mSN1, and MlST8 to mTOR was examined in ARV-infected or p17-transfected Vero cells. Cells were mock-infected or infected with ARV at an MOI of 10 and transfected with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag (vector only) plasmid for 24 hours. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot analysis, and proteins were detected with the indicated antibodies. Lower panel: Data were obtained in three independent experiments, error bars indicate the means ± SD. ( C ) Upper panel: for analysis of the effect of insulin on mTORC2, cells were treated with insulin (0.2 uM) for 1 hour, followed by transfection of cells with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag plasmid for 24 hours at 37 °C. The interaction of mTOR and rictor with Rpl7 and Rpl26 was examined by co-immunoprecipitation experiments described in panel A. Lower panel: Data shown represent the mean ± SD calculated from three independent experiments. ( D ) To study whether MDM2 and PSMB6 affect the association of mTORC2 and ribosomal proteins, depletion of MDM2 and PSMB6 with shRNAs was performed. The uncropped blots with molecular weights are shown in Figs S6 and S7 .
Figure Legend Snippet: p17 deregulates mTORC2 assembly. ( A ) To study the effect of insulin on ribosomal proteins and Akt phosphorylation at S473, cells were pretreated with insulin (0.2 μm) for 1 hour, followed by transfection with pcDNA3.1-Flag-p17 for 24 hours at 37 °C. Whole cell lysates were harvested for Western blot assays with the indicated antibodies. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against values for the mock control. those at mock. The levels of indicated proteins in the mock control were considered 1-fold. ( B ) Upper panel: in co-immunoprecipitation experiments, the binding of rictor, mSN1, and MlST8 to mTOR was examined in ARV-infected or p17-transfected Vero cells. Cells were mock-infected or infected with ARV at an MOI of 10 and transfected with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag (vector only) plasmid for 24 hours. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot analysis, and proteins were detected with the indicated antibodies. Lower panel: Data were obtained in three independent experiments, error bars indicate the means ± SD. ( C ) Upper panel: for analysis of the effect of insulin on mTORC2, cells were treated with insulin (0.2 uM) for 1 hour, followed by transfection of cells with either pcDNA3.1-Flag-p17 or pcDNA3.1-Flag plasmid for 24 hours at 37 °C. The interaction of mTOR and rictor with Rpl7 and Rpl26 was examined by co-immunoprecipitation experiments described in panel A. Lower panel: Data shown represent the mean ± SD calculated from three independent experiments. ( D ) To study whether MDM2 and PSMB6 affect the association of mTORC2 and ribosomal proteins, depletion of MDM2 and PSMB6 with shRNAs was performed. The uncropped blots with molecular weights are shown in Figs S6 and S7 .

Techniques Used: Transfection, Western Blot, Activation Assay, Immunoprecipitation, Binding Assay, Infection, Plasmid Preparation, SDS Page

14) Product Images from "Luteolin Induces Apoptosis and Autophagy in Mouse Macrophage ANA-1 Cells via the Bcl-2 Pathway"

Article Title: Luteolin Induces Apoptosis and Autophagy in Mouse Macrophage ANA-1 Cells via the Bcl-2 Pathway

Journal: Journal of Immunology Research

doi: 10.1155/2018/4623919

The effects of luteolin on ERK/p38/Akt in the ANA-1 cells. (a) Representative Western blot results showing phospho-ERK1/2/ERK1/2, phospho-p38/p38, and phospho-Akt/Akt expression in the ANA-1 cells. (b) The relative expression of proteins compared with the control. ∗ p
Figure Legend Snippet: The effects of luteolin on ERK/p38/Akt in the ANA-1 cells. (a) Representative Western blot results showing phospho-ERK1/2/ERK1/2, phospho-p38/p38, and phospho-Akt/Akt expression in the ANA-1 cells. (b) The relative expression of proteins compared with the control. ∗ p

Techniques Used: Western Blot, Expressing

15) Product Images from "Phosphorylation of Eukaryotic Translation Initiation Factor 4E and Eukaryotic Translation Initiation Factor 4E-binding Protein (4EBP) and Their Upstream Signaling Components Undergo Diurnal Oscillation in the Mouse Hippocampus"

Article Title: Phosphorylation of Eukaryotic Translation Initiation Factor 4E and Eukaryotic Translation Initiation Factor 4E-binding Protein (4EBP) and Their Upstream Signaling Components Undergo Diurnal Oscillation in the Mouse Hippocampus

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.552638

p-rpS6, p-ERK1/2, p-Akt, and p-mTOR undergo diurnal oscillation. A, hippocampal lysates obtained from wild type mice at ZT4 h and ZT16 h were probed for rpS6 phosphorylation and actin levels by Western analysis. Bar graph shows the quantification of the
Figure Legend Snippet: p-rpS6, p-ERK1/2, p-Akt, and p-mTOR undergo diurnal oscillation. A, hippocampal lysates obtained from wild type mice at ZT4 h and ZT16 h were probed for rpS6 phosphorylation and actin levels by Western analysis. Bar graph shows the quantification of the

Techniques Used: Mouse Assay, Western Blot

p-eIF4E, p-ERK1/2, p-rpS6, p-4EBP1, and p-Akt display oscillations under free running conditions. A, representative double-plotted actogram of a mouse maintained in L/D and then D/D free-running conditions. The x axis represents two consecutive 24-h time
Figure Legend Snippet: p-eIF4E, p-ERK1/2, p-rpS6, p-4EBP1, and p-Akt display oscillations under free running conditions. A, representative double-plotted actogram of a mouse maintained in L/D and then D/D free-running conditions. The x axis represents two consecutive 24-h time

Techniques Used:

16) Product Images from "Akt Inhibitor Perifosine Prevents Epileptogenesis in a Rat Model of Temporal Lobe Epilepsy"

Article Title: Akt Inhibitor Perifosine Prevents Epileptogenesis in a Rat Model of Temporal Lobe Epilepsy

Journal: Neuroscience Bulletin

doi: 10.1007/s12264-017-0165-7

Perifosine pretreatment decreases Akt and S6 activation after KA-induced acute seizures. A KA-induced seizures result in activation of the mTOR pathway. Representative western blots showing that phospho-Akt (p-Akt) and phospho-S6 (p-S6) expression in the hippocampus increased within 1 h of the onset of KA-induced seizures in perifosine-pretreated (Peri + KA) and vehicle-pretreated (Veh + KA) rats. Vehicle-treated rats that did not receive KA (Veh + KA) served as an additional control. B Quantitative summary of p-Akt/Akt and p-S6/S6 at 1 h (* P
Figure Legend Snippet: Perifosine pretreatment decreases Akt and S6 activation after KA-induced acute seizures. A KA-induced seizures result in activation of the mTOR pathway. Representative western blots showing that phospho-Akt (p-Akt) and phospho-S6 (p-S6) expression in the hippocampus increased within 1 h of the onset of KA-induced seizures in perifosine-pretreated (Peri + KA) and vehicle-pretreated (Veh + KA) rats. Vehicle-treated rats that did not receive KA (Veh + KA) served as an additional control. B Quantitative summary of p-Akt/Akt and p-S6/S6 at 1 h (* P

Techniques Used: Activation Assay, Western Blot, Expressing

Perifosine pretreatment decreases Akt and S6 activation long after KA-induced seizures. A Representative blots of Akt and S6 at different time intervals after KA-induced seizures. B Quantitative analysis showing that phosphorylation of Akt was increased from 3 h to 3 weeks after the onset of KA-induced seizures. There was a significant decrease of the p-Akt/Akt ratio compared to rats without perifosine pretreatment. C Quantitative analysis showing that phosphorylation of S6 was increased from 3 h to 3 weeks after the onset of KA-induced seizures. There was a significant decrease of the p-S6t/S6 ratio compared to rats without perifosine pretreatment. * P
Figure Legend Snippet: Perifosine pretreatment decreases Akt and S6 activation long after KA-induced seizures. A Representative blots of Akt and S6 at different time intervals after KA-induced seizures. B Quantitative analysis showing that phosphorylation of Akt was increased from 3 h to 3 weeks after the onset of KA-induced seizures. There was a significant decrease of the p-Akt/Akt ratio compared to rats without perifosine pretreatment. C Quantitative analysis showing that phosphorylation of S6 was increased from 3 h to 3 weeks after the onset of KA-induced seizures. There was a significant decrease of the p-S6t/S6 ratio compared to rats without perifosine pretreatment. * P

Techniques Used: Activation Assay

17) Product Images from "Neutrophil Interactions with Epithelial Expressed ICAM-1 Enhances Intestinal Mucosal Wound Healing"

Article Title: Neutrophil Interactions with Epithelial Expressed ICAM-1 Enhances Intestinal Mucosal Wound Healing

Journal: Mucosal immunology

doi: 10.1038/mi.2015.135

PMN migration into intestinal lumens and ligation of ICAM-1 initiate reparative responses in epithelial cells PMNs migrating across epithelial layers are retained at the luminal surface through binding to ICAM-1 that is upregulated at the apical epithelial membrane when inflammation is present. Subsequently, PMN binding to ICAM-1 results in activation of Akt and β-catenin signaling leading to enhanced epithelial cell proliferation and wound healing.
Figure Legend Snippet: PMN migration into intestinal lumens and ligation of ICAM-1 initiate reparative responses in epithelial cells PMNs migrating across epithelial layers are retained at the luminal surface through binding to ICAM-1 that is upregulated at the apical epithelial membrane when inflammation is present. Subsequently, PMN binding to ICAM-1 results in activation of Akt and β-catenin signaling leading to enhanced epithelial cell proliferation and wound healing.

Techniques Used: Migration, Ligation, Binding Assay, Activation Assay

PMN engagement of ICAM-1 triggers activation of Akt and β-catenin signaling Control and IFNγ-stimulated T84 IECs grown on permeable supports were incubated with either PMN that migrated across T84 IECs (2.5×10 5 cells/well) (a), or with anti-ICAM-1 cross-linking antibodies (to specifically cluster ICAM-1), (b) . The effects of PMN apical adhesion and ICAM-1 cross-linking on Akt and β-catenin activation (increased phosphorylation) were assessed using Western blot analysis. All western blots are representative of 3–5 independent experiments. (c-d) β-catenin transcriptional activity was analyzed using the TopFlash luciferase reporter assay. SW480 IECs were transfected with the β-catenin reporter (TopFlash; full bars) or its mutated form (FopFlash; empty bars) 72h prior to the addition of PMN or Ab-mediated ICAM-1 cross-linking. Where indicated, inhibitory Abs to CD11b (Mac-1, 20μg/ml) were introduced in combination with PMNs. IECs were incubated with Akt inhibitor (Inhibitor VIII, 10μM) 30 minutes prior to addition of either PMNs or anti-ICAM-1 cross-linking Abs. PMN ligation or specific Ab-mediated engagement of ICAM-1 triggers activation of Akt and β-catenin signaling. N =3 independent experiments; ** p
Figure Legend Snippet: PMN engagement of ICAM-1 triggers activation of Akt and β-catenin signaling Control and IFNγ-stimulated T84 IECs grown on permeable supports were incubated with either PMN that migrated across T84 IECs (2.5×10 5 cells/well) (a), or with anti-ICAM-1 cross-linking antibodies (to specifically cluster ICAM-1), (b) . The effects of PMN apical adhesion and ICAM-1 cross-linking on Akt and β-catenin activation (increased phosphorylation) were assessed using Western blot analysis. All western blots are representative of 3–5 independent experiments. (c-d) β-catenin transcriptional activity was analyzed using the TopFlash luciferase reporter assay. SW480 IECs were transfected with the β-catenin reporter (TopFlash; full bars) or its mutated form (FopFlash; empty bars) 72h prior to the addition of PMN or Ab-mediated ICAM-1 cross-linking. Where indicated, inhibitory Abs to CD11b (Mac-1, 20μg/ml) were introduced in combination with PMNs. IECs were incubated with Akt inhibitor (Inhibitor VIII, 10μM) 30 minutes prior to addition of either PMNs or anti-ICAM-1 cross-linking Abs. PMN ligation or specific Ab-mediated engagement of ICAM-1 triggers activation of Akt and β-catenin signaling. N =3 independent experiments; ** p

Techniques Used: Activation Assay, Incubation, Western Blot, Activity Assay, Luciferase, Reporter Assay, Transfection, Ligation

18) Product Images from "Energy restriction and exercise modulate angiopoietins and vascular endothelial growth factor expression in the cavernous tissue of high-fat diet-fed rats"

Article Title: Energy restriction and exercise modulate angiopoietins and vascular endothelial growth factor expression in the cavernous tissue of high-fat diet-fed rats

Journal: Asian Journal of Andrology

doi: 10.1038/aja.2011.131

Western blot analysis of total eNOS, phospho-Akt and total-Akt. Representative bands obtained by Western blot analysis of total eNOS ( a ), total-Akt ( b ) and phospho-Akt ( c ) in the rat cavernous tissue of all experimental groups. The graphs below represent the semiquantitative analysis of the aforementioned proteins ( n =4 for each group). The total eNOS and total-Akt were normalized to β-actin, used as the loading control. Phospho-Akt levels were normalized to semiquantified total-Akt in each sample. Error bars represent standard deviation. C, control animal group; ER, energy-restricted animal group; EREx, energy-restricted and exercised animal group; HFD, high-fat diet-fed animal group. * P
Figure Legend Snippet: Western blot analysis of total eNOS, phospho-Akt and total-Akt. Representative bands obtained by Western blot analysis of total eNOS ( a ), total-Akt ( b ) and phospho-Akt ( c ) in the rat cavernous tissue of all experimental groups. The graphs below represent the semiquantitative analysis of the aforementioned proteins ( n =4 for each group). The total eNOS and total-Akt were normalized to β-actin, used as the loading control. Phospho-Akt levels were normalized to semiquantified total-Akt in each sample. Error bars represent standard deviation. C, control animal group; ER, energy-restricted animal group; EREx, energy-restricted and exercised animal group; HFD, high-fat diet-fed animal group. * P

Techniques Used: Western Blot, Standard Deviation

19) Product Images from "Effect of Autophagy Inhibition on the Protection of Ischemia Preconditioning against Myocardial Ischemia/Reperfusion Injury in Diabetic Rats"

Article Title: Effect of Autophagy Inhibition on the Protection of Ischemia Preconditioning against Myocardial Ischemia/Reperfusion Injury in Diabetic Rats

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.235867

Expressions of myocardial LC3-II, beclin-1, mTOR, PI3K, and P-Akt/Akt ratio measured by Western blotting in different rat groups. Data are presented as mean ± standard deviation, n = 5 in each group. C: Control group; IRI: Ischemia/reperfusion injury group; R: Rapamycin group; W: Wortmannin group; R + IPC: Rapamycin + IPC group; W + IPC: Wortmannin + IPC group. One-way analysis of variance for intergroup comparisons. * P
Figure Legend Snippet: Expressions of myocardial LC3-II, beclin-1, mTOR, PI3K, and P-Akt/Akt ratio measured by Western blotting in different rat groups. Data are presented as mean ± standard deviation, n = 5 in each group. C: Control group; IRI: Ischemia/reperfusion injury group; R: Rapamycin group; W: Wortmannin group; R + IPC: Rapamycin + IPC group; W + IPC: Wortmannin + IPC group. One-way analysis of variance for intergroup comparisons. * P

Techniques Used: Western Blot, Standard Deviation

Western blotting for myocardial expressions of LC3-II, beclin-1, mTOR, PI3K, Akt, and P-Akt in different rat groups. C: Control group; IRI: Ischemia/reperfusion injury group; R: Rapamycin group; W: Wortmannin group; R + IPC: Rapamycin + IPC group; W + IPC: Wortmannin + IPC group. PI3K: Phosphoinositide 3 - kinase; mTOR: Mammalian target of rapamycin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
Figure Legend Snippet: Western blotting for myocardial expressions of LC3-II, beclin-1, mTOR, PI3K, Akt, and P-Akt in different rat groups. C: Control group; IRI: Ischemia/reperfusion injury group; R: Rapamycin group; W: Wortmannin group; R + IPC: Rapamycin + IPC group; W + IPC: Wortmannin + IPC group. PI3K: Phosphoinositide 3 - kinase; mTOR: Mammalian target of rapamycin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Techniques Used: Western Blot

20) Product Images from "PTEN Loss Increases PD-L1 Protein Expression and Affects the Correlation between PD-L1 Expression and Clinical Parameters in Colorectal Cancer"

Article Title: PTEN Loss Increases PD-L1 Protein Expression and Affects the Correlation between PD-L1 Expression and Clinical Parameters in Colorectal Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065821

PTEN knockdown increased Akt activation both in the presence and absence of IFN-γ. The protein expression level of PTEN, Akt and phospho-Akt Ser473 were determined by Western blotting. β-actin was used to verify equal loading. Representative images are selected from performed experiments repeated in triplicates.
Figure Legend Snippet: PTEN knockdown increased Akt activation both in the presence and absence of IFN-γ. The protein expression level of PTEN, Akt and phospho-Akt Ser473 were determined by Western blotting. β-actin was used to verify equal loading. Representative images are selected from performed experiments repeated in triplicates.

Techniques Used: Activation Assay, Expressing, Western Blot

21) Product Images from "Implications of Dll4-Notch signaling activation in primary glioblastoma multiforme"

Article Title: Implications of Dll4-Notch signaling activation in primary glioblastoma multiforme

Journal: Neuro-Oncology

doi: 10.1093/neuonc/not071

Expression of Dll4, Notch1, Hey1, PTEN, p-AKT, pan-AKT, and VEGF proteins in GBM. Total protein was extracted from the operative specimens of GBM ( n = 15) and control tissues (normal brain, n = 5). The expression of Dll4, Notch1, Hey1, PTEN, p-AKT, AKT,
Figure Legend Snippet: Expression of Dll4, Notch1, Hey1, PTEN, p-AKT, pan-AKT, and VEGF proteins in GBM. Total protein was extracted from the operative specimens of GBM ( n = 15) and control tissues (normal brain, n = 5). The expression of Dll4, Notch1, Hey1, PTEN, p-AKT, AKT,

Techniques Used: Expressing

22) Product Images from "In vitro and in vivo Anti-Tumor Effects of Pan-HER Inhibitor Varlitinib on Cholangiocarcinoma Cell Lines"

Article Title: In vitro and in vivo Anti-Tumor Effects of Pan-HER Inhibitor Varlitinib on Cholangiocarcinoma Cell Lines

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S250061

Molecular mechanisms by which varlitinib suppresses CCA cell growth. KKU-214 and KKU-100 cells were exposed to varlitinib at the indicated concentrations for 24 hrs, then induced with 100 ng/mL of EGF for 30 mins, proteins expression level were determined by Western blot analysis. Notes: ( A ) The activation of EGFR and HER2. ( B ) The expression of p-EGFR and p-HER2 after normalized with total EGFR, HER2 and β-actin. ( C ) The activation the downstream signaling molecules Erk1/2 and Akt. ( D ) The expression of p-Erk1/2 and p-Akt after normalized with total Erk1/2, AKT and β-actin. Data in ( B and D ) are represented as mean±SD of three independent experiments. Asterisks indicate statistical significance (p
Figure Legend Snippet: Molecular mechanisms by which varlitinib suppresses CCA cell growth. KKU-214 and KKU-100 cells were exposed to varlitinib at the indicated concentrations for 24 hrs, then induced with 100 ng/mL of EGF for 30 mins, proteins expression level were determined by Western blot analysis. Notes: ( A ) The activation of EGFR and HER2. ( B ) The expression of p-EGFR and p-HER2 after normalized with total EGFR, HER2 and β-actin. ( C ) The activation the downstream signaling molecules Erk1/2 and Akt. ( D ) The expression of p-Erk1/2 and p-Akt after normalized with total Erk1/2, AKT and β-actin. Data in ( B and D ) are represented as mean±SD of three independent experiments. Asterisks indicate statistical significance (p

Techniques Used: Expressing, Western Blot, Activation Assay

23) Product Images from "FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression"

Article Title: FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Journal: BMC Developmental Biology

doi: 10.1186/1471-213X-11-20

Effects of SU5402, the MEK inhibitor U0126, and the PI3K inhibitor LY294002 on gene expression in the primitive streak region . A-P': Whole mount images showing mRNA expression in control (DMSO), SU5402, U0126 and LY294002 treated embryos. Arrows in C'-D' point to Hensen's node. Brackets in k
Figure Legend Snippet: Effects of SU5402, the MEK inhibitor U0126, and the PI3K inhibitor LY294002 on gene expression in the primitive streak region . A-P': Whole mount images showing mRNA expression in control (DMSO), SU5402, U0126 and LY294002 treated embryos. Arrows in C'-D' point to Hensen's node. Brackets in k"-l" indicate the preingression epiblast. Q': Western blot analysis comparing total versus phosphorylated ERK1/2 (p-ERK) and AKT (p-AKT) in the preingression epiblast and primitive streak of DMSO versus SU5402, U0126 and LY294002 treated embryos.

Techniques Used: Expressing, Western Blot

24) Product Images from "Ultramicronized palmitoylethanolamide rescues learning and memory impairments in a triple transgenic mouse model of Alzheimer’s disease by exerting anti-inflammatory and neuroprotective effects"

Article Title: Ultramicronized palmitoylethanolamide rescues learning and memory impairments in a triple transgenic mouse model of Alzheimer’s disease by exerting anti-inflammatory and neuroprotective effects

Journal: Translational Psychiatry

doi: 10.1038/s41398-017-0076-4

Um-PEA effects on AD pathology. Evaluation of protein expression in hippocampi of 6- and 12-month-old 3×Tg-AD and age-matched Non-Tg mice chronically treated with placebo (open bars) or um-PEA (black bars). ( a ) Representative western blots for APP, BACE1, and Aβ (1–42) proteins and ( b–d ) densitometric analyses normalized to β-actin used as loading controls ( N = 3, in triplicate). ( e ) Representative western blots for Akt, p[Thr308]Akt, Gsκ-3β, p[Ser9]Gsκ-3β, p[Ser396]tau, and ( f–j ) densitometric analysis normalized to β-actin used as loading control ( N = 3, in triplicate). The results are expressed as percentage of control (Non-Tg/placebo groups). The data are presented as means ± SEM. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s multiple-comparison test (* p
Figure Legend Snippet: Um-PEA effects on AD pathology. Evaluation of protein expression in hippocampi of 6- and 12-month-old 3×Tg-AD and age-matched Non-Tg mice chronically treated with placebo (open bars) or um-PEA (black bars). ( a ) Representative western blots for APP, BACE1, and Aβ (1–42) proteins and ( b–d ) densitometric analyses normalized to β-actin used as loading controls ( N = 3, in triplicate). ( e ) Representative western blots for Akt, p[Thr308]Akt, Gsκ-3β, p[Ser9]Gsκ-3β, p[Ser396]tau, and ( f–j ) densitometric analysis normalized to β-actin used as loading control ( N = 3, in triplicate). The results are expressed as percentage of control (Non-Tg/placebo groups). The data are presented as means ± SEM. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s multiple-comparison test (* p

Techniques Used: Expressing, Mouse Assay, Western Blot

25) Product Images from "Autophagy flux inhibition, G2/M cell cycle arrest and apoptosis induction by ubenimex in glioma cell lines"

Article Title: Autophagy flux inhibition, G2/M cell cycle arrest and apoptosis induction by ubenimex in glioma cell lines

Journal: Oncotarget

doi: 10.18632/oncotarget.22594

Western blot showed that high dose (1, 2 mg/ml) of ubenimex induced LC-3II/I expression, while low dose (0.5 mg/ml) inhibited it Same trend in Beclin1 expression. These results suggested well connection of ubenimex dose with autophagy, the expression of P62 was gradually decreased with increased dose of ubenimex. Akt pathways and APN were influenced by ubenimex. p-Akt-ser473 was down regulated in a dose-dependent manner. But the total Akt expression showed no difference. Ubenimex, an APN inhibitor, also had an effect on U87 and U251 cells, with a reverse trend for cleaved caspase-3 levels and significantly inhibited the rate of bcl-2/bax.
Figure Legend Snippet: Western blot showed that high dose (1, 2 mg/ml) of ubenimex induced LC-3II/I expression, while low dose (0.5 mg/ml) inhibited it Same trend in Beclin1 expression. These results suggested well connection of ubenimex dose with autophagy, the expression of P62 was gradually decreased with increased dose of ubenimex. Akt pathways and APN were influenced by ubenimex. p-Akt-ser473 was down regulated in a dose-dependent manner. But the total Akt expression showed no difference. Ubenimex, an APN inhibitor, also had an effect on U87 and U251 cells, with a reverse trend for cleaved caspase-3 levels and significantly inhibited the rate of bcl-2/bax.

Techniques Used: Western Blot, Expressing

26) Product Images from "An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion"

Article Title: An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2018.10.006

Dgk regulates dILP secretion and insulin signaling pathway activity. ( A ) Knockdown of Dgk using dIlp2-Gal4 shows elevated TAG, glucose, and glycogen phenotypes. ( B ) dIlp2-Gal4 -driven overexpression of kinase-dead Dgk but not wild-type Dgk also decreases glucose and glycogen levels. These phenotypes in ( A–B ) are similar to those seen using fru-Gal4 . ( C ) fru > Dgk RNAi increases dILP2 and dILP5 levels in the hemolymph. ( D ) Overexpression of either Dgk.V5 or Dgk G509D .V5 using fru-Gal4 does not affect hemolymph dILP2 or dILP5 levels. ( A–D ) Data is represented as percent or fold-change relative to a Gal4/+ control ± SD, n = 5. Assays were performed three times but results from only one representative assay are shown. ( E–F ) Knockdown or overexpression of Dgk with fru-Gal4 doesn't affect dIlp2 or dIlp5 transcript levels. Overexpression of Dgk G509D .V5 increases dIlp3 levels. ( G ) fru-Gal4 -driven Dgk RNAi or overexpression of Dgk G509D .V5 result in lowered insulin pathway activity as measured by p-Ser505 Akt/total Akt levels. fru > Dgk . V5 did not affect pAkt/Akt levels. Quantification is from 3 biological replicates and is normalized to a fru-Gal4/+ control ±SD. Asterisks denote p-values based on student's t-test: *p
Figure Legend Snippet: Dgk regulates dILP secretion and insulin signaling pathway activity. ( A ) Knockdown of Dgk using dIlp2-Gal4 shows elevated TAG, glucose, and glycogen phenotypes. ( B ) dIlp2-Gal4 -driven overexpression of kinase-dead Dgk but not wild-type Dgk also decreases glucose and glycogen levels. These phenotypes in ( A–B ) are similar to those seen using fru-Gal4 . ( C ) fru > Dgk RNAi increases dILP2 and dILP5 levels in the hemolymph. ( D ) Overexpression of either Dgk.V5 or Dgk G509D .V5 using fru-Gal4 does not affect hemolymph dILP2 or dILP5 levels. ( A–D ) Data is represented as percent or fold-change relative to a Gal4/+ control ± SD, n = 5. Assays were performed three times but results from only one representative assay are shown. ( E–F ) Knockdown or overexpression of Dgk with fru-Gal4 doesn't affect dIlp2 or dIlp5 transcript levels. Overexpression of Dgk G509D .V5 increases dIlp3 levels. ( G ) fru-Gal4 -driven Dgk RNAi or overexpression of Dgk G509D .V5 result in lowered insulin pathway activity as measured by p-Ser505 Akt/total Akt levels. fru > Dgk . V5 did not affect pAkt/Akt levels. Quantification is from 3 biological replicates and is normalized to a fru-Gal4/+ control ±SD. Asterisks denote p-values based on student's t-test: *p

Techniques Used: Activity Assay, Over Expression

27) Product Images from "Developmental Exposure to Di-(2-ethylhexyl) Phthalate Induces Cerebellar Granule Cell Apoptosis via the PI3K/AKT Signaling Pathway"

Article Title: Developmental Exposure to Di-(2-ethylhexyl) Phthalate Induces Cerebellar Granule Cell Apoptosis via the PI3K/AKT Signaling Pathway

Journal: Experimental Neurobiology

doi: 10.5607/en.2018.27.6.472

Di-(2-ethylhexyl) phthalate (DEHP) induce apoptosis of cerebellar granule cells (CGCs). The apoptosis induced by DEHP is associated with inhibition of the PI3K/AKT signaling pathway. Insulin-like growth factor (IGF) 1 may partially protect CGCs from apoptosis and LY294002 aggravated the apoptosis induced by DEHP.
Figure Legend Snippet: Di-(2-ethylhexyl) phthalate (DEHP) induce apoptosis of cerebellar granule cells (CGCs). The apoptosis induced by DEHP is associated with inhibition of the PI3K/AKT signaling pathway. Insulin-like growth factor (IGF) 1 may partially protect CGCs from apoptosis and LY294002 aggravated the apoptosis induced by DEHP.

Techniques Used: Inhibition

LY294002 aggravated the suppression of PI3K/AKT signaling pathway induced by MEHP. CGCs were pre-treated with 5 µM of LY294002, an inhibitor of PI-3K/AKT pathway, for 2 h followed by treatment with 250 MEHP for 24 h. The corresponding graphs the semi-quantitative measurement of PI3K (B), p-AKT (C), AKT (D), Bcl-2 (E), Bax (F), total caspase-3 (G) and cleaved caspase-3 (H). Each column represents the mean±SD. Mean expression in CGCs from each MEHP exposure group is shown as a fold-change compared to the mean expression in the control group, which was ascribed an arbitrary value of 1. With each time point, * p
Figure Legend Snippet: LY294002 aggravated the suppression of PI3K/AKT signaling pathway induced by MEHP. CGCs were pre-treated with 5 µM of LY294002, an inhibitor of PI-3K/AKT pathway, for 2 h followed by treatment with 250 MEHP for 24 h. The corresponding graphs the semi-quantitative measurement of PI3K (B), p-AKT (C), AKT (D), Bcl-2 (E), Bax (F), total caspase-3 (G) and cleaved caspase-3 (H). Each column represents the mean±SD. Mean expression in CGCs from each MEHP exposure group is shown as a fold-change compared to the mean expression in the control group, which was ascribed an arbitrary value of 1. With each time point, * p

Techniques Used: Expressing

MEHP disturbed the PI3K/AKT signaling pathway of CGCs in vitro . (A) Western blot bands represent the expression level of proteins in the PI3K/AKT/Bcl2/Bax pathway in the four MEHP exposure (0, 25, 100 and 250 µM) groups of primary CGCs. The corresponding bar graphs show the results of the semi-quantitative measurement of p-PI3K (B), PI3K (C), p-AKT (D), AKT (E), Bcl-2 (F) and Bax (G). Each bar represents the mean±SD. Mean expression in CGCs from each MEHP exposure group is shown as a fold-change compared to the mean expression in the control group that has been ascribed an arbitrary value of 1. With each time point, * p
Figure Legend Snippet: MEHP disturbed the PI3K/AKT signaling pathway of CGCs in vitro . (A) Western blot bands represent the expression level of proteins in the PI3K/AKT/Bcl2/Bax pathway in the four MEHP exposure (0, 25, 100 and 250 µM) groups of primary CGCs. The corresponding bar graphs show the results of the semi-quantitative measurement of p-PI3K (B), PI3K (C), p-AKT (D), AKT (E), Bcl-2 (F) and Bax (G). Each bar represents the mean±SD. Mean expression in CGCs from each MEHP exposure group is shown as a fold-change compared to the mean expression in the control group that has been ascribed an arbitrary value of 1. With each time point, * p

Techniques Used: In Vitro, Western Blot, Expressing

Effect of maternal DEHP exposure on PI3K/AKT signaling pathway of pups at PN 7. (A) Western blot bands represent the expression level of proteins in the PI3K/AKT/Bcl-2/Bax pathway at PN 7 for males and females. The bar graphs show the results of the semi-quantitative measurement of p-PI3K (B), PI3K (C), p-AKT (D), AKT (E), Bcl-2 (F) and Bax (G). Each bar represents mean ± standard deviation (SD). The mean expression in offspring from 0, 30, 300, and 750 mg/kg/d exposure groups is shown as a fold-change compared to the mean expression in the 0 mg/kg/d group which was ascribed an arbitrary value of 1. With each time point, * p
Figure Legend Snippet: Effect of maternal DEHP exposure on PI3K/AKT signaling pathway of pups at PN 7. (A) Western blot bands represent the expression level of proteins in the PI3K/AKT/Bcl-2/Bax pathway at PN 7 for males and females. The bar graphs show the results of the semi-quantitative measurement of p-PI3K (B), PI3K (C), p-AKT (D), AKT (E), Bcl-2 (F) and Bax (G). Each bar represents mean ± standard deviation (SD). The mean expression in offspring from 0, 30, 300, and 750 mg/kg/d exposure groups is shown as a fold-change compared to the mean expression in the 0 mg/kg/d group which was ascribed an arbitrary value of 1. With each time point, * p

Techniques Used: Western Blot, Expressing, Standard Deviation

Effect of maternal DEHP exposure on PI3K/AKT signaling pathway of pups at PN 14. (A) Western blot bands represent the expression level of proteins in PI3K/AKT/Bcl-2/Bax pathway at PN 14 for males and females. The bar graphs show the results of the semi-quantitative measurement of p-PI3K (B), PI3K (C), p-AKT (D), AKT (E), Bcl-2 (F) and Bax (G). Each bar represents mean±SD. The mean expression in offspring from the 0, 30, 300, and 750 mg/kg/d exposure groups is shown as a fold-change compared to the mean expression in the 0 mg/kg/d group which was ascribed an arbitrary value of 1. With each time point, * p
Figure Legend Snippet: Effect of maternal DEHP exposure on PI3K/AKT signaling pathway of pups at PN 14. (A) Western blot bands represent the expression level of proteins in PI3K/AKT/Bcl-2/Bax pathway at PN 14 for males and females. The bar graphs show the results of the semi-quantitative measurement of p-PI3K (B), PI3K (C), p-AKT (D), AKT (E), Bcl-2 (F) and Bax (G). Each bar represents mean±SD. The mean expression in offspring from the 0, 30, 300, and 750 mg/kg/d exposure groups is shown as a fold-change compared to the mean expression in the 0 mg/kg/d group which was ascribed an arbitrary value of 1. With each time point, * p

Techniques Used: Western Blot, Expressing

IGF1 protects CGCs from apoptosis induced by MEHP by activating the PI3K/AKT pathway. CGCs were pre-treated with 30, 50 and 100 ng/mL of insulin-like growth factor (IGF) 1, an activator of the PI3K/AKT, signaling pathway, for 2 h followed by treatment with 250 MEHP for 24 h. (A) Western blot bands represent the expression of proteins in the PI3K/AKT/Bcl-2/Bax pathway. (B~I) Each column represents the mean±SD. The mean expression in CGCs from each group is shown as a fold-change compared to the mean expression in the control group which was ascribed an arbitrary value of 1. With each time point, * p
Figure Legend Snippet: IGF1 protects CGCs from apoptosis induced by MEHP by activating the PI3K/AKT pathway. CGCs were pre-treated with 30, 50 and 100 ng/mL of insulin-like growth factor (IGF) 1, an activator of the PI3K/AKT, signaling pathway, for 2 h followed by treatment with 250 MEHP for 24 h. (A) Western blot bands represent the expression of proteins in the PI3K/AKT/Bcl-2/Bax pathway. (B~I) Each column represents the mean±SD. The mean expression in CGCs from each group is shown as a fold-change compared to the mean expression in the control group which was ascribed an arbitrary value of 1. With each time point, * p

Techniques Used: Western Blot, Expressing

28) Product Images from "Analysis of the Fibroblast Growth Factor System Reveals Alterations in a Mouse Model of Spinal Muscular Atrophy"

Article Title: Analysis of the Fibroblast Growth Factor System Reveals Alterations in a Mouse Model of Spinal Muscular Atrophy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031202

Western blot analysis of NSC34-cells after SMN-knockdown. ( A ) Phosphorylation of FGF-downstream targets Akt and ERK was analyzed in siRNA treated (si) and scrambled siRNA (scr) transfected cells. Phospho-antibodies against Akt (pAkt, S473), and ERK1/2 (pERK, T202,T204) were used to quantify changes in phosphorylation levels compared to non-phosphorylated Akt, ERK. Four independent experiments with three replications were performed. ( B ) Densitometrical measurements of SMN-bands normalized by α-tubulin showed an efficient knockdown of 20±5% in comparison to control-siRNA-transfected cells. ( C ) pAkt normalized to non-phosphorylated Akt was upregulated by a factor of 2.8±0.7. ( D ) pERK normalized to non-phospho-ERK was upregulated by a factor of 2.1±0.3. ( E ) The functional link between FGFR-1 signaling and ERK-hyperphosphorylation was analyzed by application of the specific FGFR-1 inhibitor PD173074 (50 µM). Additionally, FGF-2 was added to the medium in a final concentration of 50 ng/ml. ( F ) Densitometrical measurements of pERK normalized to non-phospho ERK revealed an upregulation of 10.6±3.1 fold in scrambled siRNA transfected cells under FGF-2 incubation compared to scrambled siRNA transfected control conditions. When transfected with SMN siRNA, the pERK level rises up to 23.7±0.6 fold change. These differences disappeared after PD173074 incubation. Bars and values represent means with standard error of mean (SEM). Significance was tested via repeated measurements two-way ANOVA (B, C, D, n = 4, ***p
Figure Legend Snippet: Western blot analysis of NSC34-cells after SMN-knockdown. ( A ) Phosphorylation of FGF-downstream targets Akt and ERK was analyzed in siRNA treated (si) and scrambled siRNA (scr) transfected cells. Phospho-antibodies against Akt (pAkt, S473), and ERK1/2 (pERK, T202,T204) were used to quantify changes in phosphorylation levels compared to non-phosphorylated Akt, ERK. Four independent experiments with three replications were performed. ( B ) Densitometrical measurements of SMN-bands normalized by α-tubulin showed an efficient knockdown of 20±5% in comparison to control-siRNA-transfected cells. ( C ) pAkt normalized to non-phosphorylated Akt was upregulated by a factor of 2.8±0.7. ( D ) pERK normalized to non-phospho-ERK was upregulated by a factor of 2.1±0.3. ( E ) The functional link between FGFR-1 signaling and ERK-hyperphosphorylation was analyzed by application of the specific FGFR-1 inhibitor PD173074 (50 µM). Additionally, FGF-2 was added to the medium in a final concentration of 50 ng/ml. ( F ) Densitometrical measurements of pERK normalized to non-phospho ERK revealed an upregulation of 10.6±3.1 fold in scrambled siRNA transfected cells under FGF-2 incubation compared to scrambled siRNA transfected control conditions. When transfected with SMN siRNA, the pERK level rises up to 23.7±0.6 fold change. These differences disappeared after PD173074 incubation. Bars and values represent means with standard error of mean (SEM). Significance was tested via repeated measurements two-way ANOVA (B, C, D, n = 4, ***p

Techniques Used: Western Blot, Transfection, Functional Assay, Concentration Assay, Incubation

29) Product Images from "Cyp1b1-deficient retinal astrocytes are more proliferative and migratory and are protected from oxidative stress and inflammation"

Article Title: Cyp1b1-deficient retinal astrocytes are more proliferative and migratory and are protected from oxidative stress and inflammation

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00021.2019

Cytochrome P450 1B1 (Cyp1b1) deficiency does not affect protein kinase B (AKT)/SRC/MAPK signaling pathways in retinal astrocytes (ACs). A : the expression levels of Akt, phospho (p)-Akt, Erk1/2, p-Erk1/2, Src, p-Src, Jnk, p-Jnk, MAPK p38 (p38), and p-p38 were determined by Western blot analysis of lysates prepared from Cyp1b1 +/+ and Cyp1b1 −/− retinal ACs. β-Actin was used as a loading control. B : quantitative assessment of the data. Please note that no significant differences were observed between Cyp1b1 +/+ and Cyp1b1 −/− ACs.
Figure Legend Snippet: Cytochrome P450 1B1 (Cyp1b1) deficiency does not affect protein kinase B (AKT)/SRC/MAPK signaling pathways in retinal astrocytes (ACs). A : the expression levels of Akt, phospho (p)-Akt, Erk1/2, p-Erk1/2, Src, p-Src, Jnk, p-Jnk, MAPK p38 (p38), and p-p38 were determined by Western blot analysis of lysates prepared from Cyp1b1 +/+ and Cyp1b1 −/− retinal ACs. β-Actin was used as a loading control. B : quantitative assessment of the data. Please note that no significant differences were observed between Cyp1b1 +/+ and Cyp1b1 −/− ACs.

Techniques Used: Expressing, Western Blot

30) Product Images from "Hepatitis B virus upregulates cellular inhibitor of apoptosis protein 2 expression via the PI3K/AKT/NF-κB signaling pathway in liver cancer"

Article Title: Hepatitis B virus upregulates cellular inhibitor of apoptosis protein 2 expression via the PI3K/AKT/NF-κB signaling pathway in liver cancer

Journal: Oncology Letters

doi: 10.3892/ol.2020.11267

HBV induces cIAP2 expression by activating PI3K/AKT signaling pathway. (A) THLE-3 cells transfected with (−2,000/+55)cIAP2-Luc were mock-infected or infected with HBV, in the presence or absence of signaling pathway inhibitors. Cells were lysed, and luciferase activity was measured 24 h later. (B) THLE-3 cells were mock-infected or infected with HBV, then cIAP2, PI3K, AKT and p-AKT expression was determined using western blot analysis. (C) THLE-3 cells co-transfected with (−2,000/+55)cIAP2-Luc and AKT siRNA or control siRNA, were infected with HBV. Cells were lysed, and luciferase activity was measured 24 h later. (D) THLE-3 cells transfected with AKT siRNA or control siRNA were infected with HBV and cIAP2, AKT, p-AKT, NF-κB and p-NF-κB expression was determined using western blot analysis. For western blot analysis, one representative result out of three is shown. Data are presented as the mean ± standard deviation of three independent experiments. *P
Figure Legend Snippet: HBV induces cIAP2 expression by activating PI3K/AKT signaling pathway. (A) THLE-3 cells transfected with (−2,000/+55)cIAP2-Luc were mock-infected or infected with HBV, in the presence or absence of signaling pathway inhibitors. Cells were lysed, and luciferase activity was measured 24 h later. (B) THLE-3 cells were mock-infected or infected with HBV, then cIAP2, PI3K, AKT and p-AKT expression was determined using western blot analysis. (C) THLE-3 cells co-transfected with (−2,000/+55)cIAP2-Luc and AKT siRNA or control siRNA, were infected with HBV. Cells were lysed, and luciferase activity was measured 24 h later. (D) THLE-3 cells transfected with AKT siRNA or control siRNA were infected with HBV and cIAP2, AKT, p-AKT, NF-κB and p-NF-κB expression was determined using western blot analysis. For western blot analysis, one representative result out of three is shown. Data are presented as the mean ± standard deviation of three independent experiments. *P

Techniques Used: Expressing, Transfection, Infection, Luciferase, Activity Assay, Western Blot, Standard Deviation

31) Product Images from "AND-34/BCAR3 differs from other NSP homologs in induction of anti-estrogen resistance, cyclin D1 promoter activation and altered breast cancer cell morphology"

Article Title: AND-34/BCAR3 differs from other NSP homologs in induction of anti-estrogen resistance, cyclin D1 promoter activation and altered breast cancer cell morphology

Journal:

doi: 10.1002/jcp.21059

Analysis of the ability of over-expressed NSP family members to activate either the small GTPases Rac and Cdc42 or Akt serine 473 phosphorylation
Figure Legend Snippet: Analysis of the ability of over-expressed NSP family members to activate either the small GTPases Rac and Cdc42 or Akt serine 473 phosphorylation

Techniques Used:

32) Product Images from "ZNF322A-mediated protein phosphorylation induces autophagosome formation through modulation of IRS1-AKT glucose uptake and HSP-elicited UPR in lung cancer"

Article Title: ZNF322A-mediated protein phosphorylation induces autophagosome formation through modulation of IRS1-AKT glucose uptake and HSP-elicited UPR in lung cancer

Journal: Journal of Biomedical Science

doi: 10.1186/s12929-020-00668-5

ZNF322A modulates glucose uptake via the IRS1/PI3K/AKT pathway in A549 lung cancer cells. a Schematic representation of the inhibitory transcription regulation of ZNF322A on PIM3. b Relative mRNA levels of ZNF322A and PIM3 were examined by RT-qPCR analysis, and normalized by GAPDH. c-d Proteins were extracted from ZNF322A-silenced (siZNF322A) A549 cells after 48 h transfection. Protein expressions of ZNF322A and PIM3 were examined by immunoblot analysis. Representative western blot (c) and associated densitometric analysis (d) for ZNF322A and PIM3 expressions in A549 lung cancer cells. e-f Protein expressions of IRS1 S1101 and AKT S473 phosphorylation were examined by immunoblot analysis. Phosphorylation levels were determined and normalized to the level of total proteins. The normalized values were compared between siRNA control (siControl) and siZNF322A groups. g-h Effect of 2-NBDG uptake upon ZNF322A perturbation in A549 lung cancer cells. Representative fluorescence microscopy images of 2-NBDG uptake ability in siRNA control (upper) and siZNF322A-silenced (bottom) A549 cells. 2-NBDG: green; DAPI: blue; scale bar: 10 μm (g). Mean fluorescence intensity (left) and number (right) of 2-NBDG in siControl and siZNF322A cells (h). ( i ) Schematic representation of the depletion of ZNF322A by siRNAs in A549 lung cancer cells transcriptionally regulates PIM3 kinase to induces IRS1 Ser1101 phosphorylation, which attenuates PI3K/AKT signaling pathway and inhibits AKT S473 , leading to glucose uptake blockade. β-actin is the internal control to normalize protein expression. The bars represent densitometric analysis of three biological replicates and the data are shown as mean ± SD. * p
Figure Legend Snippet: ZNF322A modulates glucose uptake via the IRS1/PI3K/AKT pathway in A549 lung cancer cells. a Schematic representation of the inhibitory transcription regulation of ZNF322A on PIM3. b Relative mRNA levels of ZNF322A and PIM3 were examined by RT-qPCR analysis, and normalized by GAPDH. c-d Proteins were extracted from ZNF322A-silenced (siZNF322A) A549 cells after 48 h transfection. Protein expressions of ZNF322A and PIM3 were examined by immunoblot analysis. Representative western blot (c) and associated densitometric analysis (d) for ZNF322A and PIM3 expressions in A549 lung cancer cells. e-f Protein expressions of IRS1 S1101 and AKT S473 phosphorylation were examined by immunoblot analysis. Phosphorylation levels were determined and normalized to the level of total proteins. The normalized values were compared between siRNA control (siControl) and siZNF322A groups. g-h Effect of 2-NBDG uptake upon ZNF322A perturbation in A549 lung cancer cells. Representative fluorescence microscopy images of 2-NBDG uptake ability in siRNA control (upper) and siZNF322A-silenced (bottom) A549 cells. 2-NBDG: green; DAPI: blue; scale bar: 10 μm (g). Mean fluorescence intensity (left) and number (right) of 2-NBDG in siControl and siZNF322A cells (h). ( i ) Schematic representation of the depletion of ZNF322A by siRNAs in A549 lung cancer cells transcriptionally regulates PIM3 kinase to induces IRS1 Ser1101 phosphorylation, which attenuates PI3K/AKT signaling pathway and inhibits AKT S473 , leading to glucose uptake blockade. β-actin is the internal control to normalize protein expression. The bars represent densitometric analysis of three biological replicates and the data are shown as mean ± SD. * p

Techniques Used: Quantitative RT-PCR, Transfection, Western Blot, Fluorescence, Microscopy, Expressing

33) Product Images from "VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis"

Article Title: VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

Journal: Biology Open

doi: 10.1242/bio.017434

Depletion of clathrin heavy chain disrupts VEGF-A isoform-specific programing of Akt and ERK1/2, but not p38 MAPK activation. (A) Endothelial cells were subjected to scrambled (Scr) or clathrin heavy chain (CHC17)-specific siRNA duplexes, were stimulated with VEGF-A 165 (165), VEGF-A 121 (121) or VEGF-A 145 (145; 1.25 nM) for 0 or 15 min prior to cell lysis and processing for immunoblot analysis of signal transduction. (B-E) Quantification of VEGFR2-pY1175 (B) and VEGFR2-pY1214 (C), Akt-pS473 (D) and ERK1/2-pT202/pY204 (E) levels upon VEGF-A isoform stimulation. (F) Endothelial cells were subjected to scrambled (Scr) or clathrin heavy chain (CHC17)-specific siRNA duplexes, were stimulated with either VEGF-A 165 , VEGF-A 121 or VEGF-A 145 (1.25 nM) for 15 min prior to cell lysis and processing for immunoblotting. (G) Quantification of p38 MAPK-pT180/pY182 levels upon VEGF-A isoform stimulation. Error bars indicate ±s.e.m. ( n =3). * P
Figure Legend Snippet: Depletion of clathrin heavy chain disrupts VEGF-A isoform-specific programing of Akt and ERK1/2, but not p38 MAPK activation. (A) Endothelial cells were subjected to scrambled (Scr) or clathrin heavy chain (CHC17)-specific siRNA duplexes, were stimulated with VEGF-A 165 (165), VEGF-A 121 (121) or VEGF-A 145 (145; 1.25 nM) for 0 or 15 min prior to cell lysis and processing for immunoblot analysis of signal transduction. (B-E) Quantification of VEGFR2-pY1175 (B) and VEGFR2-pY1214 (C), Akt-pS473 (D) and ERK1/2-pT202/pY204 (E) levels upon VEGF-A isoform stimulation. (F) Endothelial cells were subjected to scrambled (Scr) or clathrin heavy chain (CHC17)-specific siRNA duplexes, were stimulated with either VEGF-A 165 , VEGF-A 121 or VEGF-A 145 (1.25 nM) for 15 min prior to cell lysis and processing for immunoblotting. (G) Quantification of p38 MAPK-pT180/pY182 levels upon VEGF-A isoform stimulation. Error bars indicate ±s.e.m. ( n =3). * P

Techniques Used: Activation Assay, Lysis, Transduction

VEGF-A isoforms promote differential VEGFR2 phosphorylation and downstream signal transduction. (A) Endothelial cells were stimulated with either VEGF-A 165 , VEGF-A 121 or VEGF-A 145 (1.25 nM) for 2.5, 5, 10 or 20 min before lysis and processing for immunoblot analysis using site-specific phospho-antibodies against VEGFR2. (B,C) Quantification of VEGFR2-pY1175 (B) and VEGFR2-pY1214 (C) levels upon VEGF-A isoform stimulation. (D) Endothelial cells were stimulated with either VEGF-A 165 , VEGF-A 121 or VEGF-A 145 (1.25 nM) for 5, 15, 30 or 60 min before lysis and processing for immunoblot analysis of signal transduction. (E,F) Quantification of Akt-pS473 (E) and ERK1/2-pT202/pY204 (F) levels upon VEGF-A isoform stimulation. (G,H) Endothelial cells were seeded into cellular assays to assess endothelial cell permeability by measuring trans-endothelial electrical resistance (TEER) (G) or proliferation (H) upon control (Con) or VEGF-A 165 (165), VEGF-A 121 (121) or VEGF-A 145 (145; 1.25 nM) stimulated conditions for 4 or 24 h respectively. Error bars indicate ±s.e.m. ( n ≥4). * P
Figure Legend Snippet: VEGF-A isoforms promote differential VEGFR2 phosphorylation and downstream signal transduction. (A) Endothelial cells were stimulated with either VEGF-A 165 , VEGF-A 121 or VEGF-A 145 (1.25 nM) for 2.5, 5, 10 or 20 min before lysis and processing for immunoblot analysis using site-specific phospho-antibodies against VEGFR2. (B,C) Quantification of VEGFR2-pY1175 (B) and VEGFR2-pY1214 (C) levels upon VEGF-A isoform stimulation. (D) Endothelial cells were stimulated with either VEGF-A 165 , VEGF-A 121 or VEGF-A 145 (1.25 nM) for 5, 15, 30 or 60 min before lysis and processing for immunoblot analysis of signal transduction. (E,F) Quantification of Akt-pS473 (E) and ERK1/2-pT202/pY204 (F) levels upon VEGF-A isoform stimulation. (G,H) Endothelial cells were seeded into cellular assays to assess endothelial cell permeability by measuring trans-endothelial electrical resistance (TEER) (G) or proliferation (H) upon control (Con) or VEGF-A 165 (165), VEGF-A 121 (121) or VEGF-A 145 (145; 1.25 nM) stimulated conditions for 4 or 24 h respectively. Error bars indicate ±s.e.m. ( n ≥4). * P

Techniques Used: Transduction, Lysis, Permeability

Schematic depicting VEGF-A isoform-specific VEGFR2 trafficking and downstream signaling transduction. Upon ligand binding (1) VEGFR2 undergoes dimerization and either differential (Y1175) or comparable (Y1214) trans-autophosphorylation of specific tyrosine residues, depending on the VEGF-A isoform used. (2) This results in distinct levels of receptor ubiquitylation (3) and internalization into EEA1-positive early endosomes. (4) Differential levels of VEGF-A isoform-stimulated VEGFR2 internalization impacts on Akt and ERK1/2 activation in combination with VEGFR2-Y1175 phosphorylation. (5) From early endosomes VEGFR2 is trafficked into late-endosomes where it under goes VEGF-A isoform-specific proteolysis prior to lysosomal degradation. (6) VEGF-A isoform-specific VEGFR2 activation and receptor trafficking, mediates their individual capacities to regulate endothelial cell permeability, proliferation and blood vessel formation. Size and magnitude of arrow denotes magnitude of response; red, reduced; green, increased.
Figure Legend Snippet: Schematic depicting VEGF-A isoform-specific VEGFR2 trafficking and downstream signaling transduction. Upon ligand binding (1) VEGFR2 undergoes dimerization and either differential (Y1175) or comparable (Y1214) trans-autophosphorylation of specific tyrosine residues, depending on the VEGF-A isoform used. (2) This results in distinct levels of receptor ubiquitylation (3) and internalization into EEA1-positive early endosomes. (4) Differential levels of VEGF-A isoform-stimulated VEGFR2 internalization impacts on Akt and ERK1/2 activation in combination with VEGFR2-Y1175 phosphorylation. (5) From early endosomes VEGFR2 is trafficked into late-endosomes where it under goes VEGF-A isoform-specific proteolysis prior to lysosomal degradation. (6) VEGF-A isoform-specific VEGFR2 activation and receptor trafficking, mediates their individual capacities to regulate endothelial cell permeability, proliferation and blood vessel formation. Size and magnitude of arrow denotes magnitude of response; red, reduced; green, increased.

Techniques Used: Transduction, Ligand Binding Assay, Activation Assay, Permeability

34) Product Images from "A disease-associated Aifm1 variant induces severe myopathy in knockin mice"

Article Title: A disease-associated Aifm1 variant induces severe myopathy in knockin mice

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2018.05.002

AIF deficiency stimulates Akt and mTOR activity. (A–B) Immunoblot analyses were performed on (A) muscle and (B) cerebellar tissues from 3 to 6 month-old animals. Densitometry is relative to wt littermates and reported as mean ± SEM. Antibodies against total and phosphorylated forms of Akt, 40-kDa proline-rich Akt substrate (PRAS40), p70 S6 kinase (P70-S6K), ribosomal Protein S6 (RPS6) were used (n = 4–9 per genotype). GAPDH and actin were used as loading control (*** p
Figure Legend Snippet: AIF deficiency stimulates Akt and mTOR activity. (A–B) Immunoblot analyses were performed on (A) muscle and (B) cerebellar tissues from 3 to 6 month-old animals. Densitometry is relative to wt littermates and reported as mean ± SEM. Antibodies against total and phosphorylated forms of Akt, 40-kDa proline-rich Akt substrate (PRAS40), p70 S6 kinase (P70-S6K), ribosomal Protein S6 (RPS6) were used (n = 4–9 per genotype). GAPDH and actin were used as loading control (*** p

Techniques Used: Activity Assay

35) Product Images from "Regulatory roles of the NMDA receptor GluN3A subunit in locomotion, pain perception and cognitive functions in adult mice"

Article Title: Regulatory roles of the NMDA receptor GluN3A subunit in locomotion, pain perception and cognitive functions in adult mice

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2012.239251

Expression of LTP-related regulators in the forebrain of adult wild-type (WT) and glutamate NMDAR subunit 3A (GluN3A) knockout (KO) mice Western blotting was used to measure the protein levels of several mediators and effectors associated with LTP induction. A – C , there is no difference in the forebrain expression of Akt ( A ), cAMP response element-binding (CREB) ( A ), protein kinase C (PKC) ( B ), protein kinase A (PKA) ( B ), phospho-Akt (p-Akt) ( C ) and phospho-CREB (p-CREB) ( C ) between adult WT and GluN3A KO mice. Each group: n = 3 or 4. Protein expression was determined in forebrain tissue lysates with β-actin as the loading control.
Figure Legend Snippet: Expression of LTP-related regulators in the forebrain of adult wild-type (WT) and glutamate NMDAR subunit 3A (GluN3A) knockout (KO) mice Western blotting was used to measure the protein levels of several mediators and effectors associated with LTP induction. A – C , there is no difference in the forebrain expression of Akt ( A ), cAMP response element-binding (CREB) ( A ), protein kinase C (PKC) ( B ), protein kinase A (PKA) ( B ), phospho-Akt (p-Akt) ( C ) and phospho-CREB (p-CREB) ( C ) between adult WT and GluN3A KO mice. Each group: n = 3 or 4. Protein expression was determined in forebrain tissue lysates with β-actin as the loading control.

Techniques Used: Expressing, Knock-Out, Mouse Assay, Western Blot, Binding Assay

36) Product Images from "Aquaporin3 Is Required for FGF-2-Induced Migration of Human Breast Cancers"

Article Title: Aquaporin3 Is Required for FGF-2-Induced Migration of Human Breast Cancers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056735

FGF-2 induces AKT and ERK1/2 phosphorylation. For AKT and ERK1/2 phosphorylation, cells were treated with FGF-2 (10 ng/ml) and harvested at (2,5, 15, 30, 60 min). Phospho-AKT (p-AKT) and phospho-ERK1/2 (p-ERK1/2) were analyzed by Western blot and normalized to total AKT(t-AKT) and total-ERK1/2 (t-ERK1/2), respectively. The results represented mean ± SEM for triplicate experiments.
Figure Legend Snippet: FGF-2 induces AKT and ERK1/2 phosphorylation. For AKT and ERK1/2 phosphorylation, cells were treated with FGF-2 (10 ng/ml) and harvested at (2,5, 15, 30, 60 min). Phospho-AKT (p-AKT) and phospho-ERK1/2 (p-ERK1/2) were analyzed by Western blot and normalized to total AKT(t-AKT) and total-ERK1/2 (t-ERK1/2), respectively. The results represented mean ± SEM for triplicate experiments.

Techniques Used: Western Blot

37) Product Images from "MicroRNA-133a Suppresses Multiple Oncogenic Membrane Receptors and Cell Invasion in Non-Small Cell Lung Carcinoma"

Article Title: MicroRNA-133a Suppresses Multiple Oncogenic Membrane Receptors and Cell Invasion in Non-Small Cell Lung Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0096765

MiR-133a modulates AKT activation and suppresses IGF-1- and TGF-β-mediated AKT signaling in lung cancer cell lines. (A) 72 hours after infection with the AS2-neo (Ctl) or AS2-Neo-miR-133a viruses, the protein levels of pAKT (Ser473), AKT and β-actin were examined in CL1-5 and A549 cells. (B) 72 hours after transfection with the anti-miR-133a inhibitor, the protein levels of pAKT (Ser473), AKT and β-actin were examined in BEAS-2B cells. (C) Measurement of the cell invasion ability of BEAS-2B cells after anti-miR-133a inhibitor (100 nM) and LY294002 (50 µM) pre-treated for 48 hours. The number of invasive cells was counted 24 hours after seeding cells in a transwell containing matrigel. (D,E) AKT phosphorylation levels of stable miR-133a-expressing CL1-5 and A549 cells or control CL1-5 and A549 cells were examined after treatment with 250 ng/ml IGF-1 for 30 min (CL1-5 cells) or 2.5 min (A549 cells) (D) or treatment with 0.2 ng/ml TGF-β for 5 min (A549 cells) (E). The quantification of the immunoblot data are expressed as a pAKT (Ser473)/AKT ratio after normalization to β-actin.
Figure Legend Snippet: MiR-133a modulates AKT activation and suppresses IGF-1- and TGF-β-mediated AKT signaling in lung cancer cell lines. (A) 72 hours after infection with the AS2-neo (Ctl) or AS2-Neo-miR-133a viruses, the protein levels of pAKT (Ser473), AKT and β-actin were examined in CL1-5 and A549 cells. (B) 72 hours after transfection with the anti-miR-133a inhibitor, the protein levels of pAKT (Ser473), AKT and β-actin were examined in BEAS-2B cells. (C) Measurement of the cell invasion ability of BEAS-2B cells after anti-miR-133a inhibitor (100 nM) and LY294002 (50 µM) pre-treated for 48 hours. The number of invasive cells was counted 24 hours after seeding cells in a transwell containing matrigel. (D,E) AKT phosphorylation levels of stable miR-133a-expressing CL1-5 and A549 cells or control CL1-5 and A549 cells were examined after treatment with 250 ng/ml IGF-1 for 30 min (CL1-5 cells) or 2.5 min (A549 cells) (D) or treatment with 0.2 ng/ml TGF-β for 5 min (A549 cells) (E). The quantification of the immunoblot data are expressed as a pAKT (Ser473)/AKT ratio after normalization to β-actin.

Techniques Used: Activation Assay, Infection, CTL Assay, Transfection, Expressing

IGF-1R and TGFBR1 mediate cell invasion through AKT signaling in lung cancer cell lines. (A) Measurement of the protein levels of receptors CL1-5 or A549 cell transfected with siEGFR, siIGF-1R or siTGFBR1. (B) Measurement of the cell invasion ability of CL1-5 or A549 cells transfected with siEGFR, siIGF-1R or siTGFBR1 for 48 hours. The number of invasive cells was counted 20 hours after the transfected cells were seeded and was presented relative to invasion of cells transfected with siCtl. (C) Measurement of the cell proliferation ability of CL1-5 or A549 cells transfected with siEGFR, siIGF-1R or siTGFBR1 for 72 hours. (D) Representative immunoblots show protein levels of pAKT (Ser473), AKT and β-actin in these cells.
Figure Legend Snippet: IGF-1R and TGFBR1 mediate cell invasion through AKT signaling in lung cancer cell lines. (A) Measurement of the protein levels of receptors CL1-5 or A549 cell transfected with siEGFR, siIGF-1R or siTGFBR1. (B) Measurement of the cell invasion ability of CL1-5 or A549 cells transfected with siEGFR, siIGF-1R or siTGFBR1 for 48 hours. The number of invasive cells was counted 20 hours after the transfected cells were seeded and was presented relative to invasion of cells transfected with siCtl. (C) Measurement of the cell proliferation ability of CL1-5 or A549 cells transfected with siEGFR, siIGF-1R or siTGFBR1 for 72 hours. (D) Representative immunoblots show protein levels of pAKT (Ser473), AKT and β-actin in these cells.

Techniques Used: Transfection, Western Blot

38) Product Images from "The reverse-mode NCX1 activity inhibitor KB-R7943 promotes prostate cancer cell death by activating the JNK pathway and blocking autophagic flux"

Article Title: The reverse-mode NCX1 activity inhibitor KB-R7943 promotes prostate cancer cell death by activating the JNK pathway and blocking autophagic flux

Journal: Oncotarget

doi: 10.18632/oncotarget.9806

KB-R7943 induced autophagosome accumulation by downregulating the PI3K/AKT/m-TOR pathway and upregulating the JNK pathway ( A ) KB-R7943 inhibited Akt phosphorylation. PC3 cells were treated with full medium or 30 μM KB-R7943 for 24 h. Densitometric quantitation of p-Akt and Akt normalized to GAPDH is shown. ( B ) KB-R7943 inhibited mTOR phosphorylation. PC3 cells were treated with full or serum-starved media or the indicated concentrations of KB-R7943 for 24 h. Densitometric quantitation of p-mTOR and mTOR normalized to GAPDH is shown. ( C and D ) Wortmannin and 3-MA reduced KB-R7943-induced LC3-II levels. PC3 cells were pretreated with Wortmannin (100 nM) or 3-MA (5 mM) for 2 h followed by treatment with full or serum-starved media or 30 μM KB-R7943 for 24 h in the presence of Wortmannin (100 nM) or 3-MA (5 mM). Densitometric quantitation of LC3-II and P62 normalized to GAPDH is shown. ( E ) eGFP-LC3-PC3 cells were pretreated with Wortmannin (100 nM) for 2 h followed by treatment with full or serum-starved media or with 30 μM KB-R7943 for 24 h in the presence of Wortmannin (100 nM). ( F ) Western blot analysis of p-ERK, ERK, p-JNK, JNK, p-p38, p38, GRP-78, IRE1, and GAPDH levels in cells treated with full or serum-starved media or with 30 μM KB-R7943 for 24 h. ( G ) Western blot analysis of p-JNK, JNK, LC3-II, p62, and GAPDH levels in cells treated with full or serum-starved media or with 30 μM KB-R7943 in the presence or absence of 10 μM SP600125 for 24 h. The data are shown as means ± SD, n = 3. *P
Figure Legend Snippet: KB-R7943 induced autophagosome accumulation by downregulating the PI3K/AKT/m-TOR pathway and upregulating the JNK pathway ( A ) KB-R7943 inhibited Akt phosphorylation. PC3 cells were treated with full medium or 30 μM KB-R7943 for 24 h. Densitometric quantitation of p-Akt and Akt normalized to GAPDH is shown. ( B ) KB-R7943 inhibited mTOR phosphorylation. PC3 cells were treated with full or serum-starved media or the indicated concentrations of KB-R7943 for 24 h. Densitometric quantitation of p-mTOR and mTOR normalized to GAPDH is shown. ( C and D ) Wortmannin and 3-MA reduced KB-R7943-induced LC3-II levels. PC3 cells were pretreated with Wortmannin (100 nM) or 3-MA (5 mM) for 2 h followed by treatment with full or serum-starved media or 30 μM KB-R7943 for 24 h in the presence of Wortmannin (100 nM) or 3-MA (5 mM). Densitometric quantitation of LC3-II and P62 normalized to GAPDH is shown. ( E ) eGFP-LC3-PC3 cells were pretreated with Wortmannin (100 nM) for 2 h followed by treatment with full or serum-starved media or with 30 μM KB-R7943 for 24 h in the presence of Wortmannin (100 nM). ( F ) Western blot analysis of p-ERK, ERK, p-JNK, JNK, p-p38, p38, GRP-78, IRE1, and GAPDH levels in cells treated with full or serum-starved media or with 30 μM KB-R7943 for 24 h. ( G ) Western blot analysis of p-JNK, JNK, LC3-II, p62, and GAPDH levels in cells treated with full or serum-starved media or with 30 μM KB-R7943 in the presence or absence of 10 μM SP600125 for 24 h. The data are shown as means ± SD, n = 3. *P

Techniques Used: Quantitation Assay, Western Blot

39) Product Images from "FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling"

Article Title: FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling

Journal: Journal of molecular and cellular cardiology

doi: 10.1016/j.yjmcc.2009.10.009

Time-dependent activation of phospho-Pak1 and phospho-Akt in isolated adult rat ventricular myocytes treated with FTY720. Adult cardiac myocytes were exposed for 5 min, 10 min and 15 min. Subsequently, PAK1 and phospho-PAK1 (Thr 423) proteins were quantified
Figure Legend Snippet: Time-dependent activation of phospho-Pak1 and phospho-Akt in isolated adult rat ventricular myocytes treated with FTY720. Adult cardiac myocytes were exposed for 5 min, 10 min and 15 min. Subsequently, PAK1 and phospho-PAK1 (Thr 423) proteins were quantified

Techniques Used: Activation Assay, Isolation

Phospho-Pak1/Pak1 (A) and phospho-Akt/Akt (B) response to ischemia and follow-up reperfusion in the absence and presence of FTY720/S1P. Ex vivo hearts were either equilibrated for 20 min (lane 1), equilibrated followed by 20 min of ischemia (lane 2),
Figure Legend Snippet: Phospho-Pak1/Pak1 (A) and phospho-Akt/Akt (B) response to ischemia and follow-up reperfusion in the absence and presence of FTY720/S1P. Ex vivo hearts were either equilibrated for 20 min (lane 1), equilibrated followed by 20 min of ischemia (lane 2),

Techniques Used: Ex Vivo

PTX sensitivity of FTY720-mediated activation of Pak1 and Akt in neonatal cardiac myocytes. Neonatal myocytes were stimulated with 25 nM FTY720 for 5 min and then assayed for phosphorylation of Pak1 and Akt by Western blotting. Representative blots are
Figure Legend Snippet: PTX sensitivity of FTY720-mediated activation of Pak1 and Akt in neonatal cardiac myocytes. Neonatal myocytes were stimulated with 25 nM FTY720 for 5 min and then assayed for phosphorylation of Pak1 and Akt by Western blotting. Representative blots are

Techniques Used: Activation Assay, Western Blot

40) Product Images from "Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-β Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/β-Catenin Pathway *"

Article Title: Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-β Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/β-Catenin Pathway *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.652586

In silico prediction of ETH targets in the Akt/Wnt/β-catenin pathways. In silico molecular docking studies suggest several targets of ETH in the Akt/Wnt/β-catenin pathways. A–C are as described in the key .
Figure Legend Snippet: In silico prediction of ETH targets in the Akt/Wnt/β-catenin pathways. In silico molecular docking studies suggest several targets of ETH in the Akt/Wnt/β-catenin pathways. A–C are as described in the key .

Techniques Used: In Silico

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Western Blot:

Article Title: Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease
Article Snippet: .. The antibodies used for Western blot analysis included (a) anti-SMYD2 (sc-79084), anti-STAT3 (sc-482), anti-p65 (sc372 and sc-8008), anti-p53 (sc-6423), and anti-PTPN13 (sc-15356), which were purchased from Santa Cruz Biotechnology Inc.; (b) anti-SMYD2 (no. 9734), anti-ERK (no. 4696), anti-S6 (no. 2217), anti-Rb (no. 9309), anti-GFP (no. 2956), anti-AKT (no. 9272), and anti–lamin A/C (no. 2032), purchased from Cell Signaling Technology; and (c) the phosphorylated antibodies for STAT3-Y705 (no. 9131), p65-S536 (no. 3031), ERK-T202/Y204 (no. 9101), S6-S235/236 (no. 2211), AKT-S473 (no. 9271), and Rb-S780 (no. 9307), also purchased from Cell Signaling Technology. .. All of the anti–methylated histone antibodies, including H3K4-mono (ab8895), H3K4-di (ab7766), H3K4-tri (ab8580), H3K36-mono (ab9048), H3K36-di (ab9049), and H3K36-tri (ab9050), anti-Ki67 antibody (ab15580), anti-T7 antibody (ab9138), and anti–pan–methyl lysine antibody (ab23366) were purchased from Abcam.

Article Title: Autocrine Role of Angiopoietins during Megakaryocytic Differentiation
Article Snippet: .. In WB experiments the following antibodies were used: Tie-1 (clone8C9) was from Novus Biologicals, Tie-2 (mAb 05584) from Upstate; TPO-R (mAb AP1016), p-Stat5A/B (mAb AF4190), p-p38 (mAb AF869) and p-ERK 1/2 (mAb AF1018), are purchased from R & D Systems, Minneapolis, USA; p-AKT (mAb 9271), p38 MAP kinase (mAb 9212), ERK 1/2 (mAb 4695), STAT5 (mAb 9363) and AKT (mAb 9272) are purchased from Cell Signaling, Beverly, MA, USA). .. Colony Assay UT7-mpl cells (500 cells/ml/dish) have been plated in methylcellulose semisolid medium supplemented with various growth factors (GM-CSF or TPO or Ang-1 or Ang-2 or TPO+Ang-1 or TPO+Ang-2).

Incubation:

Article Title: Synergistic inhibition of mesothelioma cell growth by the combination of clofarabine and resveratrol involves Nrf2 downregulation
Article Snippet: .. The membranes were incubated for 2 h at room temperature with a 1:500 dilution of anti-Nrf2 (Santa Cruz Biotechnology), anti-HO-1 (Stressgen Biotechnologies), anti-p-Akt, and anti-p-Erk antibodies (Cell Signaling Technologies). .. Next, HRP-conjugated secondary antibody was applied at a dilution of 1 : 5,000 and the signal was visualized using an ECL detection kit (Santa Cruz Biotechnology).

Article Title: β-hydroxybutyrate and hydroxycarboxylic acid receptor 2 agonists activate the AKT, ERK and AMPK pathways, which are involved in bovine neutrophil chemotaxis
Article Snippet: .. The membrane was then incubated with a polyclonal ERK 1 or AKT antibody (Cell Signaling, USA) at a dilution of 1:2000, and β-actin HRP (Santa Cruz Biotechnology, USA) was also used. .. The densitometry of the protein bands was analyzed by using ImageJ 1.51j8 software.

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    Cell Signaling Technology Inc rabbit anti human pan 4ebp1
    PRE, W3, and W6 mTORc1 signaling marker differences between LOW and HIGH responders. (A–D) Phosphorylated (p-) mTORc1 targets. There were no significant cluster effects or cluster × time interactions for said markers. p-mTOR and <t>p-4EBP1</t> demonstrated significant time effects. (E–H) Total (pan) mTOR, p70s6k, 4EBP1, and AMPKα. Again, there were no significant cluster effects or cluster × time interactions for said markers, although there were significant time effects for pan mTOR, pan p70s6k, and pan AMPKα. Values presented in line graphs are mean (standard deviation) values. (I) Representative Western blot images for a low and high responder.
    Rabbit Anti Human Pan 4ebp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc rabbit anti akt
    Loss of FlnB induces Cdk1 activity changes through β1 <t>integrin-Pi3k/Akt</t> pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p
    Rabbit Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc rabbit anti phosphorylated ser505 akt
    Dgk regulates dILP secretion and insulin signaling pathway activity. ( A ) Knockdown of Dgk using dIlp2-Gal4 shows elevated TAG, glucose, and glycogen phenotypes. ( B ) dIlp2-Gal4 -driven overexpression of kinase-dead Dgk but not wild-type Dgk also decreases glucose and glycogen levels. These phenotypes in ( A–B ) are similar to those seen using fru-Gal4 . ( C ) fru > Dgk RNAi increases dILP2 and dILP5 levels in the hemolymph. ( D ) Overexpression of either Dgk.V5 or Dgk G509D .V5 using fru-Gal4 does not affect hemolymph dILP2 or dILP5 levels. ( A–D ) Data is represented as percent or fold-change relative to a Gal4/+ control ± SD, n = 5. Assays were performed three times but results from only one representative assay are shown. ( E–F ) Knockdown or overexpression of Dgk with fru-Gal4 doesn't affect dIlp2 or dIlp5 transcript levels. Overexpression of Dgk G509D .V5 increases dIlp3 levels. ( G ) fru-Gal4 -driven Dgk RNAi or overexpression of Dgk G509D .V5 result in lowered insulin pathway activity as measured by <t>p-Ser505</t> <t>Akt/total</t> Akt levels. fru > Dgk . V5 did not affect pAkt/Akt levels. Quantification is from 3 biological replicates and is normalized to a fru-Gal4/+ control ±SD. Asterisks denote p-values based on student's t-test: *p
    Rabbit Anti Phosphorylated Ser505 Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PRE, W3, and W6 mTORc1 signaling marker differences between LOW and HIGH responders. (A–D) Phosphorylated (p-) mTORc1 targets. There were no significant cluster effects or cluster × time interactions for said markers. p-mTOR and p-4EBP1 demonstrated significant time effects. (E–H) Total (pan) mTOR, p70s6k, 4EBP1, and AMPKα. Again, there were no significant cluster effects or cluster × time interactions for said markers, although there were significant time effects for pan mTOR, pan p70s6k, and pan AMPKα. Values presented in line graphs are mean (standard deviation) values. (I) Representative Western blot images for a low and high responder.

    Journal: Frontiers in Physiology

    Article Title: Pre-training Skeletal Muscle Fiber Size and Predominant Fiber Type Best Predict Hypertrophic Responses to 6 Weeks of Resistance Training in Previously Trained Young Men

    doi: 10.3389/fphys.2019.00297

    Figure Lengend Snippet: PRE, W3, and W6 mTORc1 signaling marker differences between LOW and HIGH responders. (A–D) Phosphorylated (p-) mTORc1 targets. There were no significant cluster effects or cluster × time interactions for said markers. p-mTOR and p-4EBP1 demonstrated significant time effects. (E–H) Total (pan) mTOR, p70s6k, 4EBP1, and AMPKα. Again, there were no significant cluster effects or cluster × time interactions for said markers, although there were significant time effects for pan mTOR, pan p70s6k, and pan AMPKα. Values presented in line graphs are mean (standard deviation) values. (I) Representative Western blot images for a low and high responder.

    Article Snippet: Rabbit anti-human phospho-p70s6k (Thr389) (1:1,000; catalog #: 9234; Cell Signaling), rabbit anti-human pan p70s6k (1:1,000; catalog #: 2708; Cell Signaling), rabbit anti-human phospho-4EBP1 (Thr37/46) (1:1,000; catalog #: 2855; Cell Signaling), rabbit anti-human pan 4EBP1 (1:1,000; catalog #: 9644; Cell Signaling), rabbit anti-human phospho-mTOR (Ser2448) (1:1,000; catalog #: 2971; Cell Signaling), rabbit anti-human pan mTOR (1:1,000; catalog #: 2972; Cell Signaling), rabbit anti-human phospho-AMPKα (Thr172) (1:1,000; catalog #: 2535; Cell Signaling), rabbit anti-human pan AMPKα (1:1,000; catalog #: 2532; Cell Signaling), rabbit anti-human androgen receptor (1:1,000; catalog #: 5153; Cell Signaling) and rabbit anti-human ubiquitin (1:1,000; catalog #: 3933; Cell Signaling) were incubated with membranes overnight at 4°C in TBST with 5% bovine serum albumin (BSA).

    Techniques: Marker, Standard Deviation, Western Blot

    Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

    Journal: PLoS ONE

    Article Title: Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

    doi: 10.1371/journal.pone.0089352

    Figure Lengend Snippet: Loss of FlnB induces Cdk1 activity changes through β1 integrin-Pi3k/Akt pathway. (A, B) Immunostaining of total and phospho-β1 integrin (pS785)(postnatal day 1 radius). Phospho-β1 integrin (pS785) levels are down-regulated in FlnB knockout chondrocytes (arrows) (B). (C, E) Western blotting results show that Pi3k(p85 subunit) and phospho-Akt(pS473), as well as phospho-Cdk1(pY15) are down-regulated in FlnB knockdown (Bsh) ATDC5 cells. Total Akt levels are not changed. Total β1 integrin levels are up-regulated but phospho-β1 integrsin (pS785) are down-regulated. Results are quantified in (E). (D, F) β1 integrin activation (Itgb1) in ATDC5 cells regulates Pi3k/Akt and Cdk1 activation. Pretreatment of ATCD5 cells with fibronectin and laminin I but not collagen (col) induces up-regulation of total β1 integrin levels but down-regulation of phospho-β1 integrin (pS785) levels. Pi3k, pAkt and Cdk1(pY15) levels are down-regulated by fibronectin and laminin I. Total Akt levels are not changed. ATDC5 cells are incubated in the presence of extracellular matrix molecules: fibronectin, laminin, and collagen, which serve as ligands for the β1 integrin receptor, and activation of the downstream pathways are assessed by Western blot analyses. Con = control, Col = collagen, Fib = fibronectin, and Lam = laminin. (G) Pretreatment of ATDC5 cells with Akt inhibitor VIII decreases Akt(pS473), Cdk1(pY15) and Sox9 levels, but increases protein levels of hypertrophic markers such as Runx2 and Col0a1. * = p

    Article Snippet: The primary antibodies (for immunostaining and some also for western blotting) were: rabbit anti-FlnA monoclonal antibody (1∶300, Cat.# 2242, Epitomics, Burlingame, CA, USA); rabbit anti-FlnB polyclonal antibody (Gifted by Dr. Kao, CWRU); mouse anti-Col2a1 (Cat.# Ab3092, ABCAM, USA); rabbit anti-Col10a1 (kindly gifted by Dr. Horton and Dr. Lunstrum, Shriners Hospital for Children, Portland, OR, USA; ); rabbit anti-Pthr1 (Cat.# Ab75150, ABCAM, USA);rabbit anti-Ihh (Cat.# sc-13088, Santa Cruz); rabbit anti-Runx2 (Cat.# sc-10758, Santa Cruz); rabbit anti-Sox9 pab (1∶300, AB5535, Millipore); rabbit anti-Sox9 pab (O9-1, gift of Professor Dr. Michael Wegner, Institute of Biochemistry, Friedrich-Alexander-University, Erlangen-Nurnberg, Germany); rat anti-BrdU (1∶150, Cat.# MCA2060, AbD Serotec, Raleigh, NC, USA); rabbit anti-Ki-67 mab (1∶200, Cat.# 4203, Epitomics); rabbit anti-PH3 pab (1∶250, Cat:# 06-570, Millipore, Billerica, MA, USA); mouse and rabbit anti-Wee1 (1∶50, Cat.# sc-5285 and sc-325, Santa Cruz); rabbit anti-Pkmyt1 (1∶100, Cat.# 3303, Epitomics); anti-pan 14-3-3 (Santa Cruz, sc-629); mouse and rabbit anti-cyclin B1 (1∶100, Cat.# sc-245 and sc-752, Santa Cruz); mouse anti-Cdc20 (1∶100, Cat.# sc-13162, Santa Cruz); mouse and rabbit anti-cdc25c (1∶100, Cat.# sc-55513 and sc-327, Santa Cruz); rabbit-anti-Cdk1 (Cat.# PC25,Calbiochem, San Diego, CA, USA); mouse anti-Cdk1(pY15) (Cat.# BD612306, BD, Franklin Lakes, NJ USA); rabbit anti-Pi3k (p85 subunit alpha, Cat#: 1675, Epitomics); rabbit anti-Akt (phospho-S473, Cat# 4060, Cell Signaling); rabbit anti-Erk1/2 (phospho-T202/Y204, Cat.# 4370, Cell Signaling); rat anti-β1 integrin (Cat.# mab1997, Millipore); rabbit anti- β1 integrin(pS785) (Cat.# OPA1-03177, Affinity BioReagents).

    Techniques: Activity Assay, Immunostaining, Knock-Out, Western Blot, Activation Assay, Incubation, Laser Capture Microdissection

    Reduction in phosphorylation/activation of Akt by GT1b . (A) Phosphorylation of Akt was detected by immunoblotting with phospho-Akt (Ser473) and phospho-Akt (Thr308) antibody in cultures treated with 20 μg/ml GT1b at indicated time points. (B) The histogram shows quantification of phospho-Akt levels. (C-F) The activity of Akt was measured using immunoprecipitated Akt, and then it was mixed with the GSK-3α/β fusion protein (1 μg/assay). Phosphorylated GSK-3α/β was detected by immunoblotting with phospho-GSK-3α/β antibody. (D,F) The histograms show quantification of phospho-GSK-3α/β levels of C and E, respectively. The values represent the mean ± SEM of four to five separate experiments; *P

    Journal: BMC Neuroscience

    Article Title: GT1b-induced neurotoxicity is mediated by the Akt/GSK-3/tau signaling pathway but not caspase-3 in mesencephalic dopaminergic neurons

    doi: 10.1186/1471-2202-11-74

    Figure Lengend Snippet: Reduction in phosphorylation/activation of Akt by GT1b . (A) Phosphorylation of Akt was detected by immunoblotting with phospho-Akt (Ser473) and phospho-Akt (Thr308) antibody in cultures treated with 20 μg/ml GT1b at indicated time points. (B) The histogram shows quantification of phospho-Akt levels. (C-F) The activity of Akt was measured using immunoprecipitated Akt, and then it was mixed with the GSK-3α/β fusion protein (1 μg/assay). Phosphorylated GSK-3α/β was detected by immunoblotting with phospho-GSK-3α/β antibody. (D,F) The histograms show quantification of phospho-GSK-3α/β levels of C and E, respectively. The values represent the mean ± SEM of four to five separate experiments; *P

    Article Snippet: Equivalent amounts of Akt were immunoprecipitated by rabbit-Akt antibody prebound to protein A-agarose beads, and kinase assays were carried out according to the manufacturer's instruction manual of the Akt kinase Assay Kit (Cell Signaling Technology).

    Techniques: Activation Assay, Activity Assay, Immunoprecipitation

    Dgk regulates dILP secretion and insulin signaling pathway activity. ( A ) Knockdown of Dgk using dIlp2-Gal4 shows elevated TAG, glucose, and glycogen phenotypes. ( B ) dIlp2-Gal4 -driven overexpression of kinase-dead Dgk but not wild-type Dgk also decreases glucose and glycogen levels. These phenotypes in ( A–B ) are similar to those seen using fru-Gal4 . ( C ) fru > Dgk RNAi increases dILP2 and dILP5 levels in the hemolymph. ( D ) Overexpression of either Dgk.V5 or Dgk G509D .V5 using fru-Gal4 does not affect hemolymph dILP2 or dILP5 levels. ( A–D ) Data is represented as percent or fold-change relative to a Gal4/+ control ± SD, n = 5. Assays were performed three times but results from only one representative assay are shown. ( E–F ) Knockdown or overexpression of Dgk with fru-Gal4 doesn't affect dIlp2 or dIlp5 transcript levels. Overexpression of Dgk G509D .V5 increases dIlp3 levels. ( G ) fru-Gal4 -driven Dgk RNAi or overexpression of Dgk G509D .V5 result in lowered insulin pathway activity as measured by p-Ser505 Akt/total Akt levels. fru > Dgk . V5 did not affect pAkt/Akt levels. Quantification is from 3 biological replicates and is normalized to a fru-Gal4/+ control ±SD. Asterisks denote p-values based on student's t-test: *p

    Journal: Molecular Metabolism

    Article Title: An in vivo screen for neuronal genes involved in obesity identifies Diacylglycerol kinase as a regulator of insulin secretion

    doi: 10.1016/j.molmet.2018.10.006

    Figure Lengend Snippet: Dgk regulates dILP secretion and insulin signaling pathway activity. ( A ) Knockdown of Dgk using dIlp2-Gal4 shows elevated TAG, glucose, and glycogen phenotypes. ( B ) dIlp2-Gal4 -driven overexpression of kinase-dead Dgk but not wild-type Dgk also decreases glucose and glycogen levels. These phenotypes in ( A–B ) are similar to those seen using fru-Gal4 . ( C ) fru > Dgk RNAi increases dILP2 and dILP5 levels in the hemolymph. ( D ) Overexpression of either Dgk.V5 or Dgk G509D .V5 using fru-Gal4 does not affect hemolymph dILP2 or dILP5 levels. ( A–D ) Data is represented as percent or fold-change relative to a Gal4/+ control ± SD, n = 5. Assays were performed three times but results from only one representative assay are shown. ( E–F ) Knockdown or overexpression of Dgk with fru-Gal4 doesn't affect dIlp2 or dIlp5 transcript levels. Overexpression of Dgk G509D .V5 increases dIlp3 levels. ( G ) fru-Gal4 -driven Dgk RNAi or overexpression of Dgk G509D .V5 result in lowered insulin pathway activity as measured by p-Ser505 Akt/total Akt levels. fru > Dgk . V5 did not affect pAkt/Akt levels. Quantification is from 3 biological replicates and is normalized to a fru-Gal4/+ control ±SD. Asterisks denote p-values based on student's t-test: *p

    Article Snippet: Primary antibodies used: mouse anti-V5 (1:2000, Invitrogen), rabbit anti-Akt (1:500, Cell Signalling Technology), rabbit anti-phosphorylated Ser505 Akt (1:1000, Cell Signalling Technology) and mouse anti-actin (1:4000, Abcam).

    Techniques: Activity Assay, Over Expression